TW202330027A - Anti-cd47 antibody formulation - Google Patents

Abstract

The present invention relates to an anti-CD47 antibody formulation, a preparation method therefor and the use thereof.

Description

Anti-CD-47 antibody preparation

The present invention relates to anti-CD47 antibody formulations, methods of preparing such formulations and uses of such formulations.

CD47 (differentiation group 47) was first identified as a tumor antigen in human ovarian cancer in the 1980s. CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-related antigen, and MER6, is a multiplex membrane receptor belonging to the immunoglobulin superfamily with one immunoglobulin-like domain and five A transmembrane region that serves as a ligand for signal regulatory protein α (SIRPα) and binds to the V-like domain at the NH ₂ terminus of SIRPα. SIRPα is mainly expressed in bone marrow cells, including macrophages, granulocytes, dendritic cells (DCs), mast cells and their precursor cells, including hematopoietic stem cells. CD47 on normal cells binds to SIRPα on macrophages, releasing a “don’t eat me” signal and inhibiting the phagocytosis of macrophages, which is an important mechanism for macrophages in the innate immune system to identify self and non-self. CD47 is widely expressed in human tumor cells and tissues, including acute myeloid leukemia (AML), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM) ), bladder cancer, and other solid tumors. Tumor cells bind to SIRPα on the surface of macrophages through highly expressed CD47, evading macrophage phagocytosis and promoting tumor growth. The CD47 immune checkpoint is considered a potentially effective and widely applicable target for tumor immunotherapy. Currently, a variety of specific blockers targeting the interaction between CD47 and SIRPα have been developed. A number of ongoing preclinical and clinical trials involve drugs for the treatment of diffuse large B lymphocytic lymphoma, acute myeloid leukemia, and advanced solid tumors, including anti-CD47 antibodies and SIRPα fusion proteins. These drugs are administered alone or in combination with other anti-tumor treatments. Take the anti-CD47 antibody Hu5F9 developed by Forty Seven as an example. In a phase I clinical trial to evaluate the effect of Hu5F9 in treating 22 lymphoma patients, the combination of Hu5F9 and rituximab resulted in 50% improvement in the effectiveness of Hu5F9 in treating 22 lymphoma patients. Patients who failed to respond to tuximab alone experienced objective responses. In clinical data published in 2019, Hu5F9 combined with azacitidine achieved a complete remission rate of 36% in the treatment of 14 patients with relapsed/refractory acute myeloid leukemia, and 11 patients with myelosuppressive syndrome. The response rate of combined use can reach 55%.

As with any protein therapeutic, antibodies are subject to physical and chemical instabilities such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation, and shearing during manufacturing or storage. When high concentrations of antibody are included in the formulation, it is more difficult to maintain antibody physical and chemical stability. Therefore, it is important to determine optimal formulation conditions to improve the stability of antibodies during production, storage, and use.

Therefore, the present invention provides a pharmaceutical preparation containing a high concentration of anti-CD47 antibody or antigen-binding fragment thereof. In particular, the antibody or antigen-binding fragment thereof has high anti-tumor activity and does not cause significant red blood cell agglutination reaction. , to meet more clinical needs, and pharmaceutical preparations containing the antibody or its antigen-binding fragment show good stability under a variety of physical stress conditions that simulate the production or transportation of pharmaceuticals.

The present invention provides pharmaceutical preparations containing anti-CD47 antibodies or antigen-binding fragments thereof, especially stable high-concentration antibody liquid preparations.

In one aspect, the invention provides a pharmaceutical formulation having a pH value of about 5.0 to about 8.5, or about 5.5 to about 7.0, comprising (i) an anti-CD47 antibody or antigen-binding fragment thereof; (ii) a buffer, ( iii) stabilizers, and (iv) surfactants.

In some embodiments, pharmaceutical formulations of the invention comprise a buffer that is a histamine buffer or a citrate buffer. In some embodiments, wherein the histidine salt buffer is histidine-histidine hydrochloride buffer. In some embodiments, wherein the citrate buffer is citric acid-sodium citrate buffer.

In some embodiments, the concentration of the buffer is from about 10mM to about 80mM, preferably from about 20mM to about 50mM, for example, about 20mM, about 30mM, about 40mM, about 50mM, about 60mM or about 70mM.

In some embodiments, the pharmaceutical formulation of the present invention includes a stabilizer selected from at least one selected from the group consisting of sugars, amino acids, and polyols.

In some embodiments, the stabilizer includes at least one amino acid, such as arginine, preferably arginine hydrochloride.

In some embodiments, wherein the stabilizer consists of an amino acid, preferably arginine hydrochloride.

In some embodiments, the sugar is preferably sucrose.

In some embodiments, the stabilizer consists of an amino acid and a sugar, preferably arginine hydrochloride and sucrose.

In some embodiments, the concentration of the amino acid is from about 1 mg/mL to about 100 mg/mL, such as from about 5 mg/mL to about 40 mg/mL.

In some embodiments, the concentration of the sugar is from about 10 mg/mL to about 100 mg/mL, preferably from about 20 mg/mL to about 80 mg/mL.

In some embodiments, the (mass) concentration ratio of the amino acid and sugar is about 1:1 to about 1:15, such as about 1:1, about 1:2, about 1:3, about 1:4 , about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14 Or about 1:15.

In some embodiments, the pharmaceutical formulation of the present invention contains a surfactant selected from polysorbate surfactants, preferably polysorbate-80 or polysorbate-20.

In some embodiments, the concentration of the surfactant is about 0.002% (w/v) to about 0.5% (w/v), preferably about 0.01% (w/v) to about 0.05% (w /v), for example, about 0.01% (w/v), about 0.02% (w/v) or about 0.03% (w/v), preferably about 0.02% (w/v).

In some embodiments, pharmaceutical formulations of the invention comprise an anti-CD47 antibody or antigen-binding fragment thereof at a concentration of about 10-150 mg/mL, preferably about 40-120 mg/mL, for example, about 20 mg/mL, about 50 mg/mL, about 60 mg/mL, about 80 mg/mL, about 100 mg/mL, or about 110 mg/mL.

In some embodiments, the pH of pharmaceutical formulations of the present invention is from about 5.5 to about 7.5, from about 5.5 to about 7.0, for example from about 6.0 to about 7.0, preferably from about 6.0 to about 6.5, more preferably about 6.5.

In one aspect, the invention provides a pharmaceutical formulation comprising:

(i) About 10-150 mg/mL of anti-CD47 antibody or antigen-binding fragment thereof;

(ii) about 10-80mM citrate buffer or histamine buffer;

(iii) about 5-40 mg/mL arginine hydrochloride and about 0-80 mg/mL sucrose; and

(iv) from about 0.01% (w/v) to about 0.05% (w/v) polysorbate-80;

The pH value is about 6.0-7.0.

In one aspect, the invention provides a pharmaceutical formulation comprising:

(i) About 40-120 mg/mL of anti-CD47 antibody or antigen-binding fragment thereof;

(ii) about 20-80mM citrate buffer or histamine buffer;

(iii) About 5-40 mg/mL arginine hydrochloride and 20-80 mg/mL sucrose, wherein the mass concentration ratio of arginine hydrochloride and sucrose is 1:1 to 1:15,

(iv) from about 0.01% (w/v) to about 0.05% (w/v) polysorbate-80;

The pH value is about 6.0-7.0.

In one aspect, the invention provides a pharmaceutical formulation comprising:

(i) About 40-120 mg/mL of anti-CD47 antibody or antigen-binding fragment thereof;

(ii) about 20-80mM citrate buffer;

(iii) about 5-40 mg/mL arginine hydrochloride, and

(iv) from about 0.01% (w/v) to about 0.05% (w/v) polysorbate-80;

The pH value is about 6.0-7.0.

In some embodiments, the pharmaceutical preparation of the present invention is in liquid form, which further contains a vehicle, which includes but is not limited to water, such as water for injection, bacteriostatic water for injection (BWFI) or double-distilled water or at least two thereof. A combination of the above.

In one aspect, the present invention also provides a pharmaceutical preparation in a freeze-dried form, which is prepared by freeze-drying the pharmaceutical preparation of the present invention as described above.

In one aspect, the present invention also provides a reconstituted pharmaceutical preparation, which is prepared by reconstituting the pharmaceutical preparation in lyophilized form according to the present invention with a reconstitution medium.

In some embodiments, the reconstitution medium includes, but is not limited to, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), and glucose solution (e.g., 5% (w/v) glucose) at least one.

In some embodiments, the reconstituted pharmaceutical preparation of the present invention further includes a solvent, which includes but is not limited to water, such as water for injection, bacteriostatic water for injection (BWFI) or double distilled water or a combination of two or more thereof. .

In some embodiments, pharmaceutical formulations of the invention (including liquid form pharmaceutical formulations, lyophilized form pharmaceutical formulations, or reconstituted pharmaceutical formulations) are injectable or infusible liquid form formulations.

In one aspect, the invention also provides an article of manufacture comprising a container containing a pharmaceutical formulation of the invention (including a pharmaceutical formulation in liquid form, a pharmaceutical formulation in lyophilized form, or a reconstituted pharmaceutical formulation).

The anti-CD47 antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical preparation of the present invention can be any anti-CD47 antibody or antigen-binding fragment thereof.

Preferably, the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical preparation of the present invention has high anti-tumor activity and does not cause significant red blood cell agglutination reaction to meet more clinical needs.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises one to three selected from HCDR1, HCDR2, and HCDR3 of a heavy chain variable region (VH), wherein the VH comprises SEQ ID NOs: 1, 3, The amino acid sequence represented by 5, 6 or 7.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises one to three selected from the group consisting of heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, the HCDR1 comprising the amine group set forth in SEQ ID NO: 11 acid sequence, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 12, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 13 or 17 or 21.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises one to three selected from LCDR1, LCDR2 and LCDR3 of a light chain variable region (VL), wherein the VL comprises SEQ ID NOs 2, 4, The amino acid sequence shown in 8, 9 or 10.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises one to three selected from the group consisting of light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, the LCDR1 comprising the amine shown in SEQ ID NO: 14 The LCDR2 includes the amino acid sequence shown in SEQ ID NO: 15 or 18 or 22, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 16.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises three CDRs of the heavy chain variable domain (VH), HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable domain (VL), That is, LCDR1, LCDR2 and LCDR3, wherein the VH includes the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL includes the amino acid sequence shown in SEQ ID NO: 2, 4, 8, 9 or 10. The amino acid sequence shown.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises three CDRs of the heavy chain variable domain (VH), HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable domain (VL), That is, LCDR1, LCDR2 and LCDR3; where the VH and VL are selected from:

(1) VH comprising the amino acid sequence shown in SEQ ID NO: 1, and VL comprising the amino acid sequence shown in SEQ ID NO: 2;

(2) VH comprising the amino acid sequence shown in SEQ ID NO: 3, and VL comprising the amino acid sequence shown in SEQ ID NO: 4;

(3) VH comprising the amino acid sequence shown in SEQ ID NO: 5, and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9 or 10;

(4) VH comprising the amino acid sequence shown in SEQ ID NO: 6, and VL comprising the amino acid sequence shown in SEQ ID NO: 9 or 10; or

(5) VH comprising the amino acid sequence represented by SEQ ID NO: 7, and VL comprising the amino acid sequence represented by SEQ ID NO: 8, 9 or 10.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3 and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, wherein the HCDR1 comprises SEQ ID The amino acid sequence shown in NO: 11, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 12, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 13 or 17 or 21, LCDR1 includes SEQ ID NO : the amino acid sequence shown in SEQ ID NO: 14, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 15 or 18 or 22, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 16.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3 and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, wherein the HCDR1 comprises SEQ ID The amino acid sequence shown in NO: 11, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 17, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 14, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 includes SEQ ID NO : The amino acid sequence shown in 16.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence corresponding to SEQ ID NO: 1, 3, 5, 6 or 7 Amino acid sequences that are identical or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL), wherein the VL comprises an amino acid sequence corresponding to SEQ ID NO: 2, 4, 8, 9 or 10 Amino acid sequences that are identical or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the same sequence as SEQ ID NO: 1, 3, 5, 6 Or an amino acid sequence that is identical to the amino acid sequence shown in 7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. , and the VL contains an amino acid sequence identical to or at least 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 8, 9 or 10 , an amino acid sequence with 97%, 98% or 99% sequence identity.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises an amino acid corresponding to SEQ ID NO: 7 Sequence identity or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and the VL contains the same sequence as SEQ ID NO： The amino acid sequences shown in 8 are identical or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are selected from:

(1) VH comprising the amino acid sequence shown in SEQ ID NO: 1, and VL comprising the amino acid sequence shown in SEQ ID NO: 2;

(2) VH comprising the amino acid sequence shown in SEQ ID NO: 3, and VL comprising the amino acid sequence shown in SEQ ID NO: 4;

(3) VH comprising the amino acid sequence shown in SEQ ID NO: 5, and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9 or 10;

(4) VH comprising the amino acid sequence shown in SEQ ID NO: 6, and VL comprising the amino acid sequence shown in SEQ ID NO: 9 or 10; or

(5) VH comprising the amino acid sequence represented by SEQ ID NO: 7, and VL comprising the amino acid sequence represented by SEQ ID NO: 8, 9 or 10.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 1 and the VL contains the amino acid sequence shown in SEQ ID NO:2.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 3 and the VL contains the amino acid sequence shown in SEQ ID NO:4.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 5 And the VL contains the amino acid sequence shown in SEQ ID NO:8.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 5 and the VL contains the amino acid sequence shown in SEQ ID NO: 9.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 5 And the VL contains the amino acid sequence shown in SEQ ID NO:10.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 6 and the VL contains the amino acid sequence shown in SEQ ID NO: 9.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 6 And the VL contains the amino acid sequence shown in SEQ ID NO:10.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 7 And the VL contains the amino acid sequence shown in SEQ ID NO:8.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL contains the amino acid sequence of SEQ ID NO:9.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 7 and the VL contains the amino acid sequence shown in SEQ ID NO:10.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain constant region, such as a human IgG1 constant region, a human IgG4 constant region, a human IgG4P constant region, or a human IgG1TM constant region. The human IgG4P constant region of the invention is a mutated human IgG4 having the amino acid substitution of S228P (according to EU numbering). In some embodiments, the human IgGlTM constant region is a mutated human IgGl constant region having amino acid substitutions of L234F, L235E and P331S (according to EU numbering).

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof comprises a light chain constant region, such as a human kappa or lambda constant region.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, or a humanized antibody. In some embodiments, at least part of the framework sequences of the anti-CD47 antibodies or antigen-binding fragments thereof of the invention are human consensus framework sequences. In one embodiment, the anti-CD47 antibody or antigen-binding fragment thereof is a full length antibody, a single domain antibody such as VHH, Fab, Fab', Fab'-SH, (Fab') ₂ , a single chain antibody such as scFv, Fv, dAb (domain antibody) or bi(poly)specific antibodies.

On the other hand, the anti-CD47 antibody contained in the pharmaceutical preparation of the invention can be replaced by an immunoconjugate or immunofusion comprising an anti-CD47 antibody or a fragment thereof.

In some embodiments, the pharmaceutical formulation is an aqueous pharmaceutical formulation. The concentration of antibody present in the formulation is determined taking into account, for example, the desired dosage volume and mode of administration. Medicine of the present invention The formulation contains high concentrations of anti-CD47 antibodies. In some embodiments, the anti-CD47 antibody concentration is about 10 mg/mL to about 150 mg/mL, such as about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg /mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, or about 150 mg/mL, and includes All values in between. In some embodiments, the antibody concentration is about 50 mg/mL or about 80 mg/mL.

On the other hand, the invention also provides pharmaceutical formulations (including liquid form pharmaceutical formulations, lyophilized form pharmaceutical formulations, or reconstituted pharmaceutical formulations) described herein for treating or preventing CD47-related diseases.

In another aspect, the invention provides a method of treating or preventing a CD47-related disease, the method comprising administering to a subject an effective amount of a pharmaceutical formulation (including a liquid form pharmaceutical formulation, a lyophilized form of a pharmaceutical formulation, or a pharmaceutical formulation described herein). reconstituted pharmaceutical formulations).

In another aspect, the invention also provides the use of a pharmaceutical formulation (including a liquid form pharmaceutical formulation, a lyophilized form of a pharmaceutical formulation, or a reconstituted pharmaceutical formulation) as described herein for the treatment or prevention of CD47-related diseases.

On the other hand, the present invention also provides the use of anti-CD47 antibodies or antigen-binding fragments thereof for preparing pharmaceutical preparations of the present invention (including liquid form pharmaceutical preparations, freeze-dried form pharmaceutical preparations, or reconstituted pharmaceutical preparations), The pharmaceutical preparation is used to treat or prevent CD47-related diseases.

In some embodiments, the CD47-related diseases include various blood cancers and solid tumors, including but not limited to bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, (malignant) melanoma, leukemia, Fibroids, leiomyosarcoma, glioma, glioblastoma Blastoma, myeloma, endometrial cancer, kidney cancer, (benign) birthmark tumor, prostate cancer, thyroid cancer, cervical cancer, gastric cancer, liver cancer.

On the other hand, the present invention also provides the use of the pharmaceutical preparations described herein (including liquid form pharmaceutical preparations, lyophilized form pharmaceutical preparations, or reconstituted pharmaceutical preparations) in the preparation of medicaments for the treatment or prevention of CD47-related diseases. .

In one embodiment, the CD47-related disease is preferably cancer, for example, the cancer includes blood cancers and solid tumors, such as bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, (malignant) melanoma, Leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, endometrial cancer, kidney cancer, (benign) birthmark tumor, prostate cancer, thyroid cancer, cervical cancer, stomach cancer, liver cancer, colon cancer, Ovarian cancer, urothelial cancer, etc.

The pharmaceutical preparation of the present invention can also be combined with other therapeutic agents or treatment methods to treat or prevent CD47-related diseases.

The invention also encompasses any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof applies to any and all anti-CD47 antibodies or fragments thereof, methods and uses of the invention described herein.

Figure 1 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Raji cells.

Figure 2 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Toledo cells.

Figure 3 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of REC-1 cells.

Figure 4 shows the activity of antibody HMA02h14-48 in blocking the interaction of human CD47 with SIRPα.

Figure 5 shows the effect of antibody HMA02h14-48 on human MΦ phagocytic Raji cells.

Figure 6 shows the effect of antibody HMA02h14-48 on human MΦ phagocytosis of Toledo cells.

Figure 7 shows the effect of antibody HMA02h14-48 on human MΦ phagocytosis of REC-1 cells.

Figure 8 shows the effect of antibody HMA02h14-48 on human MΦ phagocytosis of HL-60 cells.

Figure 9 shows the effect of antibody HMA02h14-48 on the agglutination activity of red blood cells in vitro.

Figure 10 shows the ability of antibody HMA02h14-48 to bind to CD47 on the surface of human red blood cells.

Figure 11 shows the inhibition of Toledo tumor growth by Hu5F9 and HMA02h14-48.

Figure 12 shows the inhibition of REC-1 tumor growth by Hu5F9 and HMA02h14-48.

definition

Unless otherwise stated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

In order to make the present invention easier to understand, certain scientific and technical terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this document, the scientific and technical terms used herein have the meanings commonly understood by those with ordinary skill in the technical field to which this invention belongs. For definitions and terms in this field, professionals may specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein (including within the scope of the claims), the singular form includes its corresponding plural form, unless the context clearly dictates otherwise.

The term "about" means a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system . For example, "about" may mean within 1 or greater than 1 standard deviation according to practice in the art. Alternatively, "about" may refer to a range of up to 5%, 10%, or 20% (i.e., ±5%, ±10%, or ±20%).

The term "and/or" when used to connect two or more options shall be understood to mean any one of the options or any two or more of the options.

As used herein, the term "comprises" or "includes" means the inclusion of that element, integer or step, but not the exclusion of any other element, integer or step. When the term “comprises” or “includes” is used herein, it also encompasses the elements, integers, or steps stated unless otherwise specified. composed situation. For example, when reference is made to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass antibody variable regions that consist of that particular sequence.

The terms "integrin-associated protein (IAP)", "CD47" when used herein refer to proteins from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice). and rat)) any native CD47, unless otherwise stated. The term encompasses "full-length" unprocessed CD47 as well as any form of CD47 or any fragment thereof resulting from intracellular processing. The term also includes naturally occurring variants of CD47, eg, splice variants or allelic variants. In some embodiments, CD47 refers to full length CD47 from human or a fragment thereof (such as a mature fragment thereof lacking a signal peptide). In some embodiments, human CD47 refers to CD47 or a fragment thereof consistent with the amino acid sequence shown in NCBI accession number NP_001768.1. In some embodiments, the term also encompasses fusion proteins comprising CD47 or fragments thereof.

"SIRPα" refers to wild-type signal regulatory protein α, or a recombinant or non-recombinant polypeptide containing the amino acid sequence of wild-type signal regulatory protein α, or a natural or naturally occurring allelic variant of signal regulatory protein α.

The term "affinity" herein refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be expressed in terms of the dissociation constant (K _D ). Examples of assays known in the art to determine binding affinity include surface plasmon resonance (eg, BIACORE) or similar techniques (eg, ForteBio).

The term "CD47-related disease" herein refers to a non-physiological condition associated with the expression or function or activity of CD47 or associated with CD47-mediated signaling activity, including but not limited to cancer. In some embodiments, the disease would benefit from blocking CD47-related signaling.

The term "signal transduction" herein refers to the biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of CD47 to its receptor, that results in the transmission of a signal from one part of a cell to another part of the cell. Generally, transmission involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions that results in signal transduction. Penultimate processes often involve nuclear events leading to changes in gene expression.

The terms "activity" or "biological activity" or the terms "biological properties" or "biological characteristics" are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, neutralization in vivo or in vitro, or Ability to antagonize CD47 activity, enhance or activate CD47 activity, _IC50 , in vivo stability of the antibody and immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., typically with non-human homologs of the targeted peptide, or with other proteins or tissues), and retention Ability to express high levels of antibody in mammalian cells. The aforementioned properties or characteristics may be observed, measured or evaluated using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokines or growth factor production and/or secretion, signal transduction and immunohistochemistry of tissue sections of different origins including human, primate or any other origin.

The term "antibody" herein refers to any form of antibody possessing the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), Polyclonal antibodies, multispecific antibodies (eg bispecific antibodies), humanized antibodies, human antibodies, chimeric antibodies, CrossMab antibodies, or camelized single domain antibodies.

The terms "whole antibody", "full length antibody" and "intact antibody" are used interchangeably herein to refer to a sugar containing at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds protein. Each heavy chain consists of a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (herein abbreviated 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 divided into hypervariable regions (called complementarity-determining regions (CDR)), with more conservative regions (called framework regions (FR)) interposed between them. "Complementarity-determining region" or "CDR region" or " CDRs" are regions of an antibody variable domain that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for Binds to an antigenic epitope. The CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. The CDRs located within the variable domain of the antibody heavy chain are sequentially called HCDR1, HCDR2, and HCDR3, and the CDRs located within the variable domain of the antibody light chain are called LCDR1, LCDR2 and LCDR3 in turn. Each VH and VL consists of three CDRs and 4 FRs, arranged in the following order from the amino end to the carboxyl end : FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in the binding of antibodies to antigens, but displays a variety of effector functions.

In a given VH or VL amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known schemes, including, for example, the Chothia scheme (Chothia et al., Canonical structures for the hypervariable regions of immunoglobulins”,Journal of Molecular Biology, 196, 901-917 (1987)); Kabat protocol (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath ) and Contact (University College London); North protocol (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)). The CDR of the anti-CD47 antibody in the present invention can Boundaries are determined based on any protocol in the art or a combination thereof and human assessment.

The light chains of antibodies can be assigned to one of two types, termed kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. The heavy chains of antibodies can be divided into 5 main different types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM, and several of these types can be further divided into subtypes. classes, such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.

"IgG form of an antibody" refers to the IgG form to which the heavy chain constant region of an antibody belongs. For example, the IgG4 form of an antibody means that its heavy chain constant region is derived from IgG4.

The term "monoclonal antibody" herein refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies composing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and target a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

The term "antigen-binding fragment" of an antibody herein includes fragments or derivatives of an antibody, generally the antigen-binding fragment includes at least a fragment of the antigen-binding region or variable region of the antibody (eg, one or more CDRs) and retains the of at least some binding properties. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ and Fv fragments; diabodies; linear antibodies; single chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments ) and multispecific antibodies. When the binding activity of the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of the antigen-binding activity of the antibody from which it was derived. Preferred binding fragments or derivatives retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding activity of the antibody from which they are derived.

An epitope is the region of an antigen that an antibody binds to. Epitopes can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of the protein.

The term "chimeric antibody" herein is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies are from different species. Typically the variable domain is obtained from an antibody from an experimental animal such as a rodent, while the constant domain sequence is obtained from a human antibody, making the resulting chimeric antibody less likely to induce an adverse immune response in human subjects than the experimental animal antibody. Lower sex.

The term "humanized antibody" herein refers to antibody forms containing sequences from human and non-human (eg, mouse, rat) antibodies. Generally speaking, humanized antibodies contain at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR ) region is the framework region of human immunoglobulin. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc). In some cases, amino acid mutations may be introduced into a humanized antibody (e.g., variable domains, framework regions, and/or constant regions (if present)), as is known to those of ordinary skill in the art. example For example, to improve certain properties of the antibody; such antibody forms still fall into the category of "humanized antibodies" in the present invention.

As is known to one of ordinary skill in the art, an antibody may have the form of a sugar chain used in the cell that produces the antibody. For example, the antibody may contain mouse glycans when produced in mice, in mouse cells, or in fusion tumors derived from mouse cells. Alternatively, the antibody may contain rat glycans if produced in rats, in rat cells, or in fusion tumors derived from rat cells.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and Fc region variants. The native sequence Fc region covers various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 /fimmu.2014.00520.). In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term "Fc region variant" or "variant Fc region" is used interchangeably herein to refer to an Fc region that contains modifications relative to the native sequence Fc region. Fc region variants of the invention are defined by the modifications of the amino acids that make them up.

As used herein, an "immunoconjugate" is an antibody conjugated to one or more other substances, including, but not limited to, cytotoxic agents or labels. An "immunofusion" is an antibody fused by covalent linkage to one or more other peptides or polypeptides.

The term "pharmaceutical preparation" refers to a preparation, usually containing the active ingredient, in a form that allows the biological activity of the active ingredient to be effective and does not contain other ingredients that would be unacceptable toxicity to the subject to whom the preparation is administered. and or consisting of pharmaceutically acceptable excipients. Preferably, such preparations are sterile.

The term "anti-CD47 antibody preparation" means a preparation comprising an anti-CD47 antibody as an active ingredient and a pharmaceutically acceptable excipient. Following this combination, the anti-CD47 antibody as the active ingredient is suitable for therapeutic or prophylactic administration to humans or non-human animals. The antibody preparations of the present invention may, for example, be prepared as a liquid preparation in an aqueous form, for example, a ready-to-use prefilled syringe, or as a lyophilized preparation by dissolving and/or suspending in a physiologically acceptable solution immediately before use. Reconstitute (i.e., reconstitute). In some embodiments, the anti-CD47 antibody formulation is in a liquid formulation.

"Pharmaceutically acceptable excipients" are those ingredients which can be reasonably administered to a subject mammal to provide an effective dose. Suitable pharmaceutical excipients are well known in the art and include, but are not limited to, buffers, stabilizers, surfactants, vehicles, etc.

A "stable" pharmaceutical formulation means that the protein (eg, antibody) in the formulation substantially maintains an acceptable degree of its physical and/or chemical stability and/or biological activity after storage under specified conditions. A variety of analytical techniques are known in the art for determining protein stability, see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs (1991) and Jones , A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Available at selected temperatures and selected storage times measurement stability. In one embodiment, the pharmaceutical formulation is stable at room temperature or at 40°C for at least 4 weeks, and/or at 2-8°C for at least 6 months, preferably at least 1 year, and more preferably at least 2 years. In one embodiment, the pharmaceutical formulation is preferably stable upon freezing (eg, to -70°C) and thawing.

After a storage period, if the preparation shows no aggregation, precipitation, turbidity and/or denaturation; or shows very little aggregation, precipitation, turbidity and/or denaturation, the antibody can be considered to "maintain its physical stability" in the preparation. Subvisible aggregates in formulations can be determined by light scattering, and SEC-HPLC can be used to determine soluble aggregates in formulations. In addition, the stability of the preparation can be indicated by visually inspecting the appearance and visible foreign matter of the preparation, or by detecting the turbidity of the preparation by the OD350nm method, or by measuring the purity of the preparation by the non-reducing CE-SDS method. An "acceptable degree" of physical stability may mean that the percentage change in the antibody monomer detected in the formulation does not exceed 10% after storage at a specific temperature for a specific time. In some embodiments, an acceptable degree of physical stability can be manifested by a percentage change value of the antibody monomer not exceeding 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, or 1%. A pharmaceutical preparation can also be considered stable if the percentage change value of the antibody monomer does not exceed 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. of.

An antibody can be considered to "maintain its chemical stability" in a formulation if the antibody in the formulation does not show significant chemical changes after a period of storage. Chemical stability can be assessed by detecting and/or quantifying chemically altered forms of the antibody. For example, charge variants of antibodies in preparations can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (icIEF). In one embodiment, the stability of a formulation is measured by determining the percent change in the charge variant of the antibody in the formulation after storage at a specific temperature for a specific time, wherein the change is greater than The smaller, the higher the stability of the preparation. An "acceptable degree" of chemical stability may mean that the percentage change in the charge variant (such as the main component or the acidic component or the basic component) in the formulation after storage at a specific temperature for a specific time does not exceed 30%, for example, 20 %. In some embodiments, an acceptable degree of chemical stability may be represented by a percentage change in the charge variant of the main component of no more than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. The percentage change value of the acidic component charge variant is 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 pharmaceutical preparation may also be considered to be Stable.

If the antibody in the preparation does not show significant changes in biological activity after a storage period, the antibody can be considered to "maintain its biological activity" in the preparation. For example, the binding between the antibody and the antigen can be detected by ELISA, FACS or BIACORE. An "acceptable level" of biological activity means that the percentage change in the biological activity of the antibody in the preparation after storage at a specific temperature for a specific time does not exceed 30%, 20%, or 10%.

The term "lyophilized preparation" refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition with a water content of less than 5%, preferably less than 3%.

w/v refers to the weight to volume ratio. Unless otherwise stated, for example, 0.01% (w/v) refers to 0.1 mg/mL.

The term "reconstituted" formulation refers to a liquid formulation obtained by dissolving and/or suspending a solid formulation (eg, a lyophilized formulation) in a physiologically acceptable solution. For this reconstitution, available reconstitution media include but are not limited to water for injection, bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), glucose solution (such as 5% (w/v) glucose).

The term "isotonic" formulation means that the formulation of interest has substantially the same osmotic pressure as human blood. Isotonic formulations will typically have an osmolality of about 250 to 350 mOsm/kg. For example, isotonicity can be measured using a vapor pressure or freezing point osmometer.

As used herein, the term "vehicle" refers to a liquid that can be used to dissolve or suspend active and inactive ingredients to form a pharmaceutically acceptable preparation. Vehicles that can be used in the present invention include, but are not limited to, water such as water for injection, bacteriostatic water for injection (BWFI), double distilled water, or combinations thereof.

The terms "carcinoma" and "cancer" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases.

In some embodiments according to the present invention, "treating" a disease or condition means ameliorating the disease or condition (ie, slowing or arresting or reducing at least one of the progression of the disease or its clinical symptoms). In other embodiments, "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discernible by the patient. In other embodiments, "treating" or "treating" refers to modulating a disease or condition physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of body parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.

In further embodiments according to the present invention, "prevention" of a disease or condition includes inhibition of the onset or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventive regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

In certain embodiments, cancer is "treated" by the methods of the present invention, and an individual is deemed to have been successfully treated if the individual exhibits one or more of the following: reduction in the number or complete disappearance of cancer cells; reduction in tumor size; inhibition or Lack of cancer cell invasion into peripheral organs includes, for example, spread of cancer into soft tissue and bone; inhibition or lack of tumor metastasis; inhibition or lack of tumor growth; relief of one or more symptoms associated with that particular cancer; reduction of morbidity and mortality; improvement of quality of life ; Reduce the tumorigenicity, tumorigenicity, or tumorigenicity of a tumor; reduce the number or frequency of cancer stem cells in a tumor; cause tumorigenic cells to differentiate into a non-tumorigenic state; or a combination of effects.

"Tumor growth inhibition" refers to any mechanism by which tumor cell growth can be inhibited. In certain embodiments, tumor cell growth is inhibited by delaying tumor cell proliferation. In certain embodiments, tumor cell growth is inhibited by halting tumor cell proliferation. In certain embodiments, tumor cell growth is inhibited by killing tumor cells. In certain embodiments, tumor cell growth is inhibited by inducing tumor cell apoptosis. In certain embodiments, tumor cell growth is inhibited by inducing tumor cell differentiation. In certain embodiments, tumor cell growth is inhibited by depriving tumor cells of nutrients. In certain embodiments, tumor cell growth is inhibited by preventing tumor cell migration. In certain embodiments, tumor cell growth is inhibited by preventing tumor cell invasion.

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 within a comparison window. "(Percent) Sequence Identity" can be calculated by comparing two optimally aligned sequences in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiply the result by 100 , to generate percent sequence identity. Optimal alignment for determining percent sequence identity can be accomplished 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 of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment within the full-length sequences being compared or within the sequence region of interest. In the present invention, as for the antibody sequence, the amino acid sequence identity percentage is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and in a preferred embodiment, performing the optimal alignment according to the Kabat numbering rule. , to be confirmed.

The term "agglutination" herein refers to the clumping of cells, and the term "hemagglutination" refers to the clumping of a specific type of cells, namely red blood cells. Therefore, hemagglutination is a type of agglutination.

The control antibody "Hu5F9" in this article is an anti-CD47 antibody in the form of IgG4P recombinantly expressed by GenScript based on the 5F9 variable region sequence disclosed in patent US2015/0183874 A1. The control antibody "SRF231" is an anti-CD47 antibody in the form of IgG4P recombinantly expressed by GenScript according to the 2.3D11 variable region sequence disclosed in patent US20180201677A1.

pharmaceutical preparations

The pharmaceutical preparations provided by the present invention are stable. Various methods are known in the art for testing the stability of antibody preparations. For example, non-reducing CE-SDS and SEC-HPLC can be used to analyze the purity of antibody preparations and evaluate the aggregation level of antibodies; capillary isoelectric focusing electrophoresis (cIEF), imaging capillary isoelectric focusing electrophoresis (icIEF) can be used ) and ion exchange chromatography (IEX), etc., to analyze charge variants in antibody preparations; it can be done by ELISA, FACS or BIACORE are used to detect the binding between antibodies and antigens and analyze the biological activity and stability of the antibodies in the preparation.

In one embodiment, high temperature storage at 40°C for at least 4 weeks, repeated freeze-thaw at -70°C/5°C, and/or storage at 2-8°C for at least 6 months (preferably at least 1 year, and more preferably at least 2 years years), the antibody preparation of the present invention is clear, slightly opalescent or has no visible foreign matter.

In one embodiment, high temperature storage at 40°C for at least 4 weeks, repeated freezing and thawing at -70°C/5°C, and/or storage at 2-8°C for at least 6 months (preferably at least 1 year, and more preferably at least 2 years years), the anti-CD47 antibody purity in the antibody preparation of the invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or above 99%, As determined by size exclusion chromatography or by non-reducing CE-SDS.

In one embodiment, high temperature storage at 40°C for at least 4 weeks, repeated freeze-thaw at -70°C/5°C, and/or storage at 2-8°C for at least 6 months (preferably at least 1 year, and more preferably at least 2 years years), at least 50%, preferably at least 55%, of the anti-CD47 antibodies in the antibody preparations of the invention are in the non-basic and non-acidic form (i.e., the main peak or main charge form), such as by imaging capillary isoelectric Determined by focused electrophoresis.

In one embodiment, high temperature storage at 40°C for at least 4 weeks, repeated freeze-thaw at -70°C/5°C, and/or storage at 2-8°C for at least 6 months (preferably at least 1 year, and more preferably at least 2 years years), the biological activity change value of the anti-CD47 antibody in the antibody preparation of the present invention does not exceed 30%, such as determining the specific binding activity of the antibody to the antigen by ELISA.

The present invention provides a stable pharmaceutical formulation comprising (i) an anti-CD47 antibody or antigen-binding fragment thereof; (ii) a buffer; (iii) a stabilizer; and (iv) a surfactant.

(I) Anti-CD47 antibody

100nM、

10nM、

5nM、

4nM、

3nM、

2nM、

1nM、

0.9nM、或

0.8nM。在一些實施方案中，該抗體結合全長人CD47或其片段(尤其是其胞外結合片段)。在一些實施方案中，抗-CD47抗體結合包含全長CD47或其片段的蛋白。在一些實施方案中，抗-CD47抗體結合細胞表面表達的CD47或其片段。 Antibodies suitable for use in the pharmaceutical formulations of the invention can be any anti-CD47 antibody. In some embodiments, an "anti-CD47 antibody" as described herein refers to an antibody that is capable of binding the CD47 protein with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting CD47. For example, the dissociation constants (KD) of anti-CD47 antibodies provided herein

100nM,

10nM,

5nM,

4nM,

3nM,

2nM,

1nM,

0.9nM, or

0.8nM. In some embodiments, the antibody binds full-length human CD47 or fragments thereof (especially extracellular binding fragments thereof). In some embodiments, anti-CD47 antibodies bind proteins comprising full-length CD47 or fragments thereof. In some embodiments, anti-CD47 antibodies bind cell surface expressed CD47 or fragments thereof.

In some embodiments, anti-CD47 antibodies are capable of blocking the interaction between CD47 and SIRPα, have high anti-tumor activity, and do not cause significant levels of red blood cell agglutination.

In some embodiments, the anti-CD47 antibody is the anti-CD47 antibody described in Chinese application 202010282924.0 (submitted on April 10, 2020). For the purposes of this application, the entire contents of this Chinese application are hereby incorporated by reference.

The precise amino acid sequence boundaries of the CDRs of the antibody variable regions of the invention can be determined using any of a number of well-known protocols (eg, Kabat, Chothia, AbM, Contact, or North). It should be noted that the boundaries of the CDRs of the variable region of the same antibody obtained based on different definition systems may be different. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different. Therefore, when referring to an antibody defined by a specific CDR sequence as defined in the invention, the scope of the antibody also encompasses antibodies whose variable region sequences comprise that specific CDR sequence. CDR sequences, however, due to the application of different schemes (such as different assignment systems or combinations), their claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention.

In some embodiments, the CDR boundaries of anti-CD47 antibody molecules described herein are determined based on the Kabat assignment system.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimal overlapping region can be determined, thereby providing the "minimum binding unit" for antigen binding. The smallest binding unit may be a subportion of a CDR. As will be apparent to one of ordinary skill in the art, the remaining residues in the CDR sequence can be determined from the structure of the antibody and protein folding. Therefore, variants of any CDR given herein are also contemplated by the present invention. In some embodiments, in one CDR variant of the anti-CD47 antibody or antigen-binding fragment thereof of the invention, the amino acid residues of the minimal binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or IMGT groups can be replaced by conserved amino acid residues.

In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof described herein comprise one to three selected from the group consisting of heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, wherein the HCDR1 comprises SEQ ID NO: The amino acid sequence of 11 is identical or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences, HCDR2 contains An amine group that is identical to the amino acid sequence of SEQ ID NO: 12 or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitution, preferably conservative substitution) compared with it acid sequence, HCDR3 contains the SEQ ID The amino acid sequence of NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21 is identical or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions) , preferably conservatively substituted) amino acid sequence.

In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof described herein comprise one to three selected from the group consisting of light chain complementarity determining region 1 (HCDR1), LCDR2 and LCDR3, wherein the LCDR1 comprises the same sequence as SEQ ID NO. : The amino acid sequence of 14 is identical or has at least 1 and no more than 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence, LCDR2 Comprises an amino acid sequence that is identical to or has at least 1 and no more than 3, 2 or 1 amino acid changes compared with SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 22 (preferably Amino acid substitution, preferably conservative substitution), LCDR3 contains an amino acid sequence that is identical to or has at least 1 and no more than 3, 2 or 1 amino groups compared with the amino acid sequence of SEQ ID NO: 16 Acid-modified (preferably amino acid substitution, preferably conservative substitution) amino acid sequence.

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof described herein also encompasses an antibody or antigen-binding fragment thereof wherein on the three CDRs of the heavy chain variable region, relative to the three CDRs specifically disclosed herein, CDRs, including a sequence of at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions), and/or in the light chain variable region Three CDRs, relative to the three CDRs specifically disclosed herein, contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequence.

In some embodiments, anti-CD47 antibodies, or antigen-binding fragments thereof, described herein also encompass antibodies or antigen-binding fragments thereof, wherein they are identical to the antibodies specifically disclosed herein. Compared with the heavy chain variable region and/or the light chain variable region, the heavy chain variable region and/or the light chain variable region has 1 or more (preferably no more than 10, more preferably no more than 6 or 5 , 4, 3, 2, 1) the amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) is composed of the amino acid sequence, preferably, the amine Base acid changes do not occur in the CDR regions.

In some embodiments, the heavy chain variable region (VH) of an anti-CD47 antibody or antigen-binding fragment thereof described herein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 6, or 7 Amino acid sequences that are identical or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, the light chain variable region (VL) of the anti-CD47 antibody or antigen-binding fragment thereof of the invention comprises an amino acid sequence identical to that selected from SEQ ID NO: 2, 4, 8, 9 or 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In one embodiment of the invention, amino acid changes described herein include amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions, such as conservative substitutions that do not significantly change the activity of the antibody.

In preferred embodiments, the amino acid changes occur in regions outside the CDRs (eg, in the FR). More preferably, the amino acid changes described in the present invention occur in a region outside the heavy chain variable region and/or outside the light chain variable region. In some embodiments, the amino acid changes occur in the heavy chain constant region and/or the light chain constant region.

In one embodiment of the invention, amino acid changes described herein include amino acid substitutions, insertions, or deletions. Preferably, the amino acids described herein are changed to amino acid substitutions, preferably conservative substitutions. In a preferred aspect, conservative substitutions are from Table A below The exemplary substituted residues shown are preferably the preferred conservative amino acid substituted residues shown in Table A.

In some embodiments, the antibodies described herein that contain amino acid changes have comparable or similar properties to the specific antibodies disclosed herein.

In some embodiments, the anti-CD47 antibodies described herein include post-translational modifications to the CDRs, light chain variable region, heavy chain variable region, light chain, or heavy chain.

In some embodiments, the anti-CD47 antibodies described herein are full-length antibodies, single domain antibodies, or bi(multi)specific antibodies. In some embodiments, anti-CD47 antibody fragments such as VHH, Fab antibody, Fab' antibody, Fab'-SH, (Fab') ₂ antibody, single chain antibody such as scFv, Fv, or dAb (domain antibody).

In some embodiments, the anti-CD47 antibodies described herein are any IgG form of antibody, such as an IgG1, IgG2, IgG3, or IgG4 form of antibody. In some embodiments, the anti-CD47 antibody is an IgG4P form of the antibody, i.e., the hinge region within the human IgG4 constant region is modified Ser228Pro (S228P, according to EU numbering) to avoid or reduce strand exchange.

In some embodiments, the Fc region of an antibody described herein may introduce one or more amino acid modifications, thereby generating Fc region variants. Fc region variants may comprise a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions, such as in Bruhns and Jönsson In an article published in Immunol Rev. 2015 Nov; 268(1): 25-51, on page 44, a number of modifications to human IgG1 are summarized to enhance or reduce its binding to FcγR and enhance or reduce the corresponding Function.

In some embodiments, the anti-CD47 antibodies described herein comprise Fc region variants that have reduced or lacking FcγR binding activity. In some embodiments, the Fc region variant has an amino acid substitution, specifically, the amino acid substitution is selected from the group consisting of an additional amino acid at position E233, L234, L235, N297, and P331 of the immunoglobulin heavy chain. replace. exist In some embodiments, the amino acid substitution of the Fc region variant is E233P, L234A, L235A, L235E, N297A, N297D, or P331S.

In some embodiments, the antibodies herein are altered to increase or decrease the extent of glycosylation of the antibody. The addition or deletion of glycosylation sites in an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites. Glycosylation can be altered to, for example, increase the affinity of the antibody for the "antigen". This modification can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This aglycosylation increases the affinity of the antibody for the antigen. Such methods are described, for example, in US Patent No. 5,426,300. When an antibody contains an Fc region, the sugars attached to it can be changed. In some applications, modifications that remove unwanted glycosylation sites are useful, such as removal of fucose modules to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

In some embodiments, the anti-CD47 antibodies described herein are cysteine-engineered antibodies, such as "thioMAbs," wherein one or more residues of the antibody are substituted with cysteine residues.

In some embodiments, the antibodies herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art. The non-protein moiety includes, but is not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinylpyrrolidone, -1,3-dioxane, poly-1,3,6-triane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n -vinylpyrrolidone)polyethylene glycol Alcohols, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the pharmaceutical formulation is an aqueous pharmaceutical formulation. The concentration of antibody present in the formulation is determined taking into account, for example, the desired dosage volume and mode of administration. Pharmaceutical formulations of the invention contain high concentrations of anti-CD47 antibodies. In some embodiments, the anti-CD47 antibody concentration is about 10 mg/mL to about 150 mg/mL, such as about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg /mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, or about 150 mg/mL, and includes All values and ranges in between. In some embodiments, the antibody concentration is about 50 mg/mL or about 80 mg/mL.

Immunoconjugates and immunofusions

In some embodiments, the anti-CD47 antibodies in pharmaceutical formulations of the invention can be replaced with immunoconjugates comprising anti-CD47-antibodies or fragments thereof, the immunoconjugates comprising any of the anti-CD47 antibodies provided herein or Its antigen-binding fragments and other substances. In one embodiment, the other substance is, for example, a cytotoxic agent, which includes any agent that is harmful to cells.

In some embodiments, the invention provides pharmaceutical formulations comprising immunofusions of any of the provided anti-CD47-antibodies or antigen-binding fragments thereof.

(II) Buffer

The term "buffer" as used herein refers to a pharmaceutical excipient used to stabilize the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, phosphate buffer, or its mixture. The buffer provided by the present invention has a concentration of about 10mM to About 80mM, preferably about 10mM to about 50mM. In one embodiment, the concentration of buffer is about 10mM, about 20mM, about 30mM, about 40mM or about 50mM, preferably about 20mM.

"Citrate buffer" is a buffer containing citrate ions. The citrate buffer may include one or more of citric acid, sodium citrate, potassium citrate, etc. In some embodiments, the citrate buffer is citric acid-sodium citrate buffer.

"Histidine buffer" refers to a buffer containing histidine ions. The histidine buffer may include one or more of histidine, histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. In some embodiments, the histidine buffer is histidine-histidine hydrochloride buffer.

Independent of the buffer used, the pH of the solution can be adjusted to about 5.0-7.0 with acids or bases known in the art, such as hydrochloric acid, acetic acid, phosphoric acid and citric acid, sodium hydroxide and potassium hydroxide. In one embodiment, the pH value is adjusted to about 5.0-7.0, about 5.0-6.5, about 5.0-6.0, about 5.5-7.0, about 6.0-7.0, or about 6.5-7.0, preferably the pH value is adjusted to about 5.0 , about 5.5, about 6.0, about 6.5, about 7.0, preferably the pH value is adjusted to about 6.5.

In some embodiments, pharmaceutical formulations of the invention comprise about 20 mM acetate buffer at a pH of about 5.0-6.5, preferably at a pH of about 5.0 or about 5.5. In one embodiment, the pharmaceutical formulation of the present invention contains about 20 mM citrate buffer (such as citric acid-sodium citrate buffer) with a pH value of about 5.0-6.5, preferably a pH value of about 6.0 or about 6.5. In one embodiment, the pharmaceutical formulation of the present invention contains about 20 mM phosphate buffer with a pH of about 5.0-6.5, preferably a pH of about 6.5. In one embodiment, the pharmaceutical formulation of the present invention contains about 20 mM histamine buffer (such as histidine-histidine hydrochloride buffer), The pH value is about 5.0-6.5, preferably the pH value is about 5.5, about 6.0 or about 6.5, and more preferably the pH value is about 6.5.

(III) Stabilizer

The term "stabilizer" as used herein means a pharmaceutical excipient used to protect the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during production, storage and use.

The pharmaceutical formulation of the present invention contains at least one stabilizer, which may be selected from the group consisting of amino acids, sugars, polyols and salts, and combinations thereof.

The term "amino acid" as used herein refers to a pharmaceutically acceptable organic molecule having an amine moiety located alpha-position to the carboxyl group. Examples of amino acids include, but are not limited to, arginine, glycine, ornithine, lysine, histamine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, Phenylalanine, tyrosine, tryptophan, methionine, serine, proline. The amino acid used in each case is preferably an L-type amino acid. Basic amino acids such as arginine, histidine or lysine are preferably used in the form of their inorganic salts (advantageously in the form of hydrochlorides, ie as amino hydrochlorides). The preferred amino acid used in the present invention is arginine, preferably arginine hydrochloride. The concentration of arginine hydrochloride used is about 1 mg/mL to about 100 mg/mL, 5 mg/mL to about 40 mg/mL, such as about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 26 mg /mL, about 32 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL or about 90 mg/mL, more preferably about 26 mg/mL.

The term "sugar" as used herein includes monosaccharides and oligosaccharides. Monosaccharides are monomeric carbohydrates that cannot be hydrolyzed by acids, including simple sugars and their derivatives (such as amino sugars). Sugar is usually used in its D configuration. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, Sugar, deoxyribose, neuramine. Oligosaccharides are carbohydrates composed of more than one monomeric sugar unit linked by branched or linear glycosidic bonds. The monomeric sugar units within an oligosaccharide can be the same or different. Depending on the number of monomer sugar units, oligosaccharides are disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, etc. In contrast to polysaccharides, monosaccharides and oligosaccharides are soluble in water. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose and raffinose. Preferred sugars for use in the present invention are sucrose and trehalose (ie, alpha, alpha-D-trehalose), most preferably sucrose. Useful trehalose is trehalose dihydrate. In some embodiments, the concentration of sugar (eg, sucrose) in the pharmaceutical formulation is from about 10 mg/mL to about 100 mg/mL, preferably from about 20 mg/mL to about 80 mg/mL, such as about 20 mg/mL, about 30 mg/mL. mL, about 50 mg/mL, about 65 mg/mL, or about 80 mg/mL.

The term "polyol" as used herein refers to pharmaceutically acceptable alcohols having more than one hydroxyl group. Suitable polyols include, but are not limited to, mannitol, sorbitol, glycerin (glycerol), dextran, arabitol, propylene glycol, polyethylene glycol, and combinations thereof. In some embodiments, the polyol (eg, mannitol, sorbitol) is present in the pharmaceutical formulation at a concentration of about 20 mg/mL to about 100 mg/mL. In some embodiments, the polyol (eg, mannitol, sorbitol) is present in the pharmaceutical formulation at a concentration of about 40 mg/mL to about 60 mg/mL, preferably about 45 mg/mL.

The "salt" contained in the pharmaceutical preparation of the present invention refers to inorganic salts, such as sodium chloride, magnesium chloride, and calcium chloride.

In some embodiments, the stabilizer in the pharmaceutical formulation of the invention is an amino acid, preferably arginine. In one embodiment, the stabilizer is a combination of amino acid and sugar. Preferably, the amino acid is arginine and the sugar is sucrose or trehalose. In one embodiment, the stabilizer is a combination of an amino acid and a polyol. Preferably, the amino acid is arginine and the polyol is sorbitol or glycerol. Lu alcohol. In one embodiment, the stabilizer is a combination of an amino acid and a salt. Preferably, the amino acid is arginine and the salt is sodium chloride.

In some embodiments, the stabilizer in the pharmaceutical formulation of the invention is arginine, wherein the concentration of arginine is from about 5 mg/mL to about 40 mg/mL, preferably about 26 mg/mL and about 32 mg/mL. In one embodiment, the stabilizer in the pharmaceutical preparation of the present invention is a combination of arginine and sucrose, wherein the mass concentration ratio of arginine and sucrose is about 1:1 to about 1:20, such as about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, John 1:13, John 1:14, John 1:15, John 1:16, John 1:17, John 1:18, John 1:19, and John 1:20.

In one embodiment, the pharmaceutical formulations of the invention are isotonic.

(IV) Surfactant

The pharmaceutical formulations of the present invention may also contain surfactants. The term "surfactant" generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress to reduce aggregation of antibodies or to minimize the formation of particulate matter in a formulation. The surfactant in the pharmaceutical preparation of the present invention may be a non-ionic surfactant such as polysorbate (e.g. polysorbate-20, polysorbate-80, etc.) or poloxamer (e.g. poloxamer- 188 etc.).

The amount of surfactant contained in the pharmaceutical formulations of the present invention may vary depending on the specific intended properties of the formulation, the specific environment, and the specific purpose for which the formulation is to be used. In some embodiments, the surfactant (e.g., polysorbate-80) is present in the pharmaceutical formulation at a concentration of about 0.002% (w/v) (i.e., 0.02 mg/mL) to about 0.5% (w/v) ( i.e., 5 mg/mL), about 0.01% (w/v) (i.e., 0.1 mg/mL) to about 0.1% (w/v) (i.e., 1 mg/mL). In some embodiments In the pharmaceutical formulation, the surfactant (e.g., polysorbate-80) is present at a concentration of about 0.01% (w/v) (i.e., 0.1 mg/mL) to about 0.05% (w/v) (i.e., 0.5 mg /mL), preferably about 0.01% (w/v) (i.e., 0.1mg/mL), about 0.02% (w/v) (i.e., 0.2mg/mL) or about 0.03% (w/v) (i.e., 0.3 mg/mL), more preferably about 0.02% (w/v) (i.e., 0.2 mg/mL).

For other reasons, other excipients may also be used in the formulations of the present invention. Such excipients include, for example, flavoring agents, antimicrobial agents, sweeteners, antistatic agents, antioxidants, alum, and the like. These and other known pharmaceutical excipients and/or additives suitable for use in the formulations of the present invention are well known in the art and are listed, for example, in "The Handbook of Pharmaceutical Excipients, 4th Edition, Rowe et al., eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)."

The pharmaceutical preparations according to the invention may also be provided in lyophilized form or in liquid form reconstituted from the lyophilized form. "Lyophilized forms" are prepared by freeze-drying methods known in the art. Lyophilisates typically have a residual moisture content of about 0.1% (w/w) to 5% (w/w) and are available as powders or physically stable cakes. "Reconstituted form" can be obtained by rapidly dissolving the lyophilisate after addition of reconstitution medium. Suitable reconstitution media include, but are not limited to, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), and glucose solution (e.g., 5% (w/v) glucose).

Preparation of formulations

Any of the anti-CD47 antibodies described herein may be produced using methods known in the art, for example, methods comprising the step of culturing a protein containing DNA encoding any of the antibodies described herein under conditions suitable for the production of such antibodies in a manner suitable for expression. Anti-CD47 antibody nucleic acid host cells (HEK293 cells or HEK293T, HEK293F, HEK293E cells modified based on HEK293 cells; CHO cells or CHO-S, CHO-dhfr-, CHO/DG44, ExpiCHO modified based on CHO cells), recover the cells culture medium, and purify the antibody using conventional purification methods.

Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599-613) describe the use of ion exchange chromatography (anionic IEX and/or cationic CEX chromatography) after the protein A capture step. Antibody three-column purification method. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies. Biotechnol Bioeng. 2008 Oct 15;101(3):553-66.) describe a two-column purification method in which weak partitioning anion exchange is used after Protein A affinity chromatography. resin.

After obtaining an antibody of sufficient purity, preparations containing the antibody can be prepared according to methods known in the art. For example, the following steps can be used for preparation: (1) After the fermentation, the fermentation broth is centrifuged and clarified to remove impurities such as cells to obtain the supernatant; (2) Affinity chromatography (for example, with specific affinity for IgG1, IgG2 and IgG4 type antibodies (Protein A column) to capture the antibody; (3) perform virus inactivation; (4) refine and purify (CEX cation exchange chromatography can generally be used) to remove impurities in the protein; (5) virus filtration (to increase the virus titer Reduced, for example, above 4log10); (6) Ultrafiltration/diafiltration (can be used to replace the protein in a preparation buffer that facilitates its stability and concentrate it to a suitable concentration for injection). See, eg, Minow B, Rogge P, Thompson K. Implementing a Fully Disposable MAb Manufacturing Facility. BioProcess Int. 10(6) 2012: 48-58.

Treatment or use

In one aspect, the invention provides methods of preventing, diagnosing, or treating CD47-related diseases in a subject. The method includes administering to a patient in need thereof an effective amount of a pharmaceutical formulation described herein.

In one aspect, the invention provides use of an anti-CD47 antibody or antigen-binding fragment thereof in the production or preparation of a pharmaceutical formulation for preventing, diagnosing, or treating a CD47-related disease in a subject.

In one aspect, the invention provides pharmaceutical preparations for preventing or treating CD47-related diseases in subjects. Pharmaceutical formulations disclosed herein may be administered against CD47-related diseases in a subject identified using standard methods.

In some embodiments, a CD47-related disease as described herein refers to a disease associated with abnormal CD47 expression, activity, and/or signaling in a subject, including but not limited to cancer. In some embodiments, in a CD47-related disease, the nucleic acid (level or amount) encoding CD47 is increased, or CD47 expression is increased, or CD47 protein levels are increased, or activity is increased, or activity signaling is enhanced.

In some embodiments, treatment of the disease would benefit from inhibiting CD47 at the nucleic acid or protein level, or from blocking the binding of CD47 to its ligands or CD47-mediated signaling.

The term "subject" or "patient" or "individual" herein includes any human or non-human animal. The term "non-human animals" includes all vertebrate animals, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like. In some embodiments, the subject may be a mammal, for example, a primate, preferably a tall Primates, eg, humans (eg, individuals suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject has a disease (eg, cancer) or is at risk of having a disease described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy treatments and/or radiation therapy.

In some embodiments, the cancer includes various hematological cancers and solid tumors, as well as metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. Cancer can be early, intermediate or late or metastatic. Such cancers include bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, endometrial cancer, kidney cancer , (benign) birthmark tumors, prostate cancer, thyroid cancer, cervical cancer, gastric cancer, liver cancer. In some embodiments, the lymphoma is selected from Burkitt lymphoma, diffuse large cell lymphoma, or mantle cell lymphoma. In some embodiments, the leukemia is promyelocytic leukemia.

The pharmaceutical preparations of the present invention may be administered by any suitable means, including oral, parenteral, intrapulmonary and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing regimens are considered herein, including, but not limited to, single or multiple dosing at various time points, bolus dosing, and pulse infusion.

In some embodiments, a pharmaceutical formulation described herein can be administered to a subject or individual in combination with another therapeutic agent to treat a disease. For example, to treat cancer, a pharmaceutical formulation described herein can be administered in combination with another anti-cancer treatment, such as chemotherapy or a different antibody treatment. In some embodiments, the therapeutic agent, such as a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a vaccine, Other antibodies, immunomodulators or other biological macromolecule drugs. In some embodiments, treatment modalities include surgery; radiation therapy, localized or focused irradiation, and the like. The above-mentioned combination therapy includes combined administration (in which two or more therapeutic agents are contained in the same or separate formulations) and separate administration, wherein the administration of the pharmaceutical formulation of the present invention can occur with additional therapeutic agents and/or adjuvants. before, at the same time and/or after the administration of the agent and/or treatment.

The "therapeutically effective amount" of an antibody will depend on, for example, the condition to be treated, the severity and course of the condition, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapies, the patient's clinical history and response to the antibody, the antibody used type, and at the discretion of the attending physician. The antibody is suitably administered to the patient in one sitting or in a series of treatments, and may be administered to the patient at any time after diagnosis. The antibodies can be administered as sole treatment or in combination with other drugs or therapies useful in treating the condition in question.

As a general recommendation, a therapeutically effective amount of antibody administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight, whether by one or multiple administrations. In some embodiments, the antibody used is, for example, administered daily from about 0.01 to about 45 mg/kg, from about 0.01 to about 40 mg/kg, from about 0.01 to about 35 mg/kg, from about 0.01 to about 30 mg/kg, from about 0.01 to about 30 mg/kg. About 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg. In some embodiments, the antibody is administered at about 15 mg/kg. However, other dosage regimens may be useful. The dose may be administered as a single dose or as multiple doses (eg 2 or 3 doses), such as infusion. The dose of antibody administered in combination therapy can be reduced compared to single therapy. The progress of this therapy is easily monitored by conventional techniques.

Products or kits

The present invention provides articles of manufacture or kits containing a container containing a pharmaceutical preparation of the present invention and optionally instructions for its use. Suitable containers include, for example, bottles, vials, bags and syringes. Containers can be made from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloys (such as stainless steel or Hastelloy). A container contains a pharmaceutical preparation and a label on or associated with the container indicates instructions for use. Articles of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with printed instructions for use. In some embodiments, the article of manufacture further includes one or more another agent (eg, a chemotherapeutic agent and an antineoplastic agent). Suitable containers for the one or more medicaments include, for example, bottles, vials, bags and syringes.

Combination products

In one aspect, the invention also provides a combination product comprising a pharmaceutical formulation of the invention, together with one or more other therapeutic agents. The combination products of the invention can be used in the treatment methods of the invention.

The invention includes any combination of the specific embodiments described herein. It should be understood that although specific content and examples have been described to illustrate preferred embodiments of the present invention, these are illustrative and exemplified only, and the present invention also encompasses those that would be obvious to those of ordinary skill in the art. Modified embodiments of the preferred embodiments of the invention. All publications, patents, and patent applications cited herein, including citations, are hereby incorporated by reference in their entirety for all purposes.

[Detailed implementation]

Preparation and activity verification of antibodies in pharmaceutical preparations of the present invention

Example 1 Preparation and screening of fusion tumor antibodies

Anti-CD47 antibodies were obtained using fusion tumor technology, and mice were immunized with the recombinant protein CD47-Fc (ACROBiosystems, Cat: CD7-H5256) with Fc-tagged human CD47 extracellular domain as the antigen. CD47-Fc recombinant protein was mixed and emulsified with complete or incomplete Freund's adjuvant (Sigma-Aldrich), and then immunized to SJL (Beijing Vitong Lever Laboratory Animal Technology Co., Ltd.) and BALB/c (Yangzhou University Medical Center) mice. . After the mice underwent one round of immunization (complete Freund's adjuvant) and two rounds of booster immunization (incomplete Freund's adjuvant), blood was collected after each booster immunization, and ELISA technology was used to detect the post-immune mouse serum and recombinant Human CD47-Fc (ACROBiosystems, Cat: CD7-H5256) protein binding activity, and the binding potency of mouse serum and CHO cells (GenScript construct) overexpressing human CD47 were detected by flow cytometry (FACS). The spleen cells of mice with high serum titers were selected and fused with the myeloma cell line SP2/0 (ATCC). Four days before fusion, the recombinant protein CD47-Fc of human CD47 extracellular domain was intraperitoneally injected into mice to enhance immunity. On the day of fusion, the mice were euthanized, and the mouse spleen cells were homogenized to obtain a single-cell suspension. Mouse spleen cells were fused (3:1) with mouse myeloma cell line SP2/0 using an electrofusion instrument. The fused cells were resuspended in medium containing HAT (hypoxanthine, aminopterin and thymidine, GIBCO, Cat: 21060016) to screen out the fusion tumor cells that successfully fused. The supernatant of fusion tumor cells was collected, and fusion tumor cells secreting antibodies that specifically bind to human CD47 were screened out through two rounds of ELISA. Subsequently, CD47-related functional screening experiments (such as binding specificity to human or cynomolgus CD47; no activity to promote erythrocyte agglutination; promotion of macrophage phagocytosis of tumor cells) were used to identify the activity of the fusion tumor secreted supernatant, and positive fusions The pure tumor strain is subjected to single or multiple rounds of sub-selection to obtain a single strain. After screening, 125G4A4 was the final selected pure fusion tumor strain.

The candidate fusion tumor cells 125G4A4 were expanded and cultured. After 7-10 days of culture, the supernatant was collected, centrifuged and filtered to remove cells and debris. The supernatant was passed through a Protein A purification column (GenScript), and then cleaned and equilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0), and then eluted with 0.1M sodium citrate (pH 3.5), and Immediately neutralize with one-ninth volume of 1 M Tris-HCl (pH 9) and then dialyze against PBS buffer. The fusion tumor antibody 125G4A4 was finally obtained for further characterization.

1.1 FACS detection of binding activity of antibodies to CHO-K1 cells overexpressing human CD47 protein

Human CD47 protein (NCBI accession number: NP_001768.1) was overexpressed in the hamster ovary cell line CHO-K1 to establish a CHO-K1 cell line overexpressing human CD47 protein. The cells were incubated with the serially diluted antibody 125G4A4 and the control antibody C0774CK230-C (i.e. Hu5F9) (the highest concentration was 300 nM, three times diluted, a total of 12 concentration points), and incubated at 4°C for 50 minutes. The cells were washed twice with ice-cold PBS, iFluor647-labeled goat anti-mouse IgG (H+L) antibody (Genscript) was added, and incubated at 4°C in the dark for 40 minutes. After the cells were washed twice with ice-cold PBS, the fluorescence signal was detected by a Calibur (BD Biosciences) flow cytometer, and a concentration-dependent curve was fitted with GraphPad based on its mean fluorescence intensity (MFI) and the EC ₅₀ was calculated. As shown in Table 1, the finally obtained fusion tumor antibody 125G4A4 has high binding activity to CHO-K1 overexpressing human CD47 protein, with an EC ₅₀ of 0.22nM.

1.2 FACS detection of antibody binding activity to CD47 on the surface of tumor cell lines

The human Burkitt lymphoma cell line Raji cells endogenously express human CD47 on the surface. The antibody 125G4A4 and the control antibody Hu5F9 were serially diluted into PBS containing 2% fetal bovine serum (FBS, Gibco, Cat: 10100147) (the highest concentration was 46.3nM, Three times dilution, a total of 8 concentration points), mix the diluted antibody with Raji cells (purchased from ATCC) (5*10 ⁵ cells per well), and incubate at 4°C for 1 hour. The cells were then washed three times with PBS containing 2% fetal bovine serum (FBS), PE-labeled mouse anti-human IgG Fc antibody (Biolegend, Cat: 409304) was added, and incubated at 4°C in the dark for 1 hour. After the cells were washed three times with PBS containing 2% fetal bovine serum (FBS), the fluorescence signal was detected by a CantoII (BD Biosciences) flow cytometer, and the concentration dependence was fitted using GraphPad according to its mean fluorescence intensity (MFI). curve and calculate EC ₅₀ . As shown in Table 1, fusion tumor antibody 125G4A4 has binding activity to Raji cells, with an EC ₅₀ of 0.84±0.02nM.

1.3 Anti-CD47 antibody blocks the interaction between human CD47 and SIRPα

The ELISA method was used to detect the ability of 125G4A4 to block the binding of human CD47 to SIRPα. Human CD47 extracellular segment and human IgG Fc-terminal recombinant protein hCD47-Fc (ACROBiosystems, Cat: CD7-H5256) were coated on a 96-well plate and incubated at 4°C overnight. After washing the plate three times with PBST (PBS containing 0.5% Tween-20), add PBST containing 1% BSA to block for 2 hours. After washing the plate three times with PBST, serially diluted antibody 125G4A4 and control antibody Hu5F9 (highest concentration 66.7 nM, three times diluted, a total of 8 concentration points) and SIRPα-His recombinant protein (ACROBiosystems, 2016) with a final concentration of 2.5 μg/ml were added. Cat: SIA-5225) and incubate at room temperature for 1 hour. Wash the plate three times with PBST, and add horseradish peroxidase-labeled goat anti-His-tag secondary antibody (CWBIO, Cat: CW0285M) to detect SIRPα captured by coated CD47. After incubating the 96-well plate at 37°C for 30 minutes, wash the plate 5 times with PBST, add TMD (Surmodics, Cat: TMBW-1000-01) chromogenic solution, and incubate in the dark for 15 minutes. The color reaction was terminated by adding 2N H ₂ SO ₄ . Read the OD ₄₅₀ on a microplate reader. The absorbance value reflects the amount of SIRPα bound to CD47. Use Graphpad to fit a concentration-dependent curve and calculate the IC ₅₀ of anti-CD47 antibody blocking the binding of CD47 to SIRPα. As shown in Table 1, 125G4A4 can effectively block the interaction between CD47 and SIRPα, with an IC ₅₀ of 3.06nM.

1.4 Detection of anti-CD47 antibody-induced agglutination activity of human red blood cells

It is known in the art that most anti-CD47 antibodies have the property of inducing red blood cell agglutination. It is generally believed that this property is closely related to side effects such as anemia that occur with therapeutic anti-CD47 antibodies. To this end, we evaluated the anti-CD47 antibodies of the present invention through in vitro erythrocyte agglutination assay to screen for antibodies without erythrocyte agglutination-promoting properties. The specific method is as follows: collect fresh human blood from healthy donors, wash it five times with PBS, dilute it into a suspension containing 10% human red blood cells, and mix the red blood cell suspension with experimental antibodies (antibody 125G4A4 and control antibody Hu5F9, the highest concentration 667nM, three times diluted, a total of 12 concentration points), mix and add to a round-bottom 96-well plate, incubate at room temperature for 16 hours, take pictures and judge the results based on the performance of the cells in the wells. If agglutination of red blood cells occurs, the cells will be spread out into a network, and a larger sheet-like cell layer will appear in the well, with a diameter larger than that of the negative control well; conversely, if agglutination does not occur, red blood cells will be deposited at the bottom of the well. , smaller dot-like cell clusters will precipitate in the wells. 125G4A4 did not have obvious erythrocyte agglutination-inducing properties in this experiment.

1.5 Detection of the effect of anti-CD47 antibodies on the phagocytosis of tumor cells by human macrophages

The flow cytometry-based assay method detects the ability of the antibody 125G4A4 of the present invention to promote macrophage phagocytosis of tumor cells. Fresh blood from healthy donors was collected, and peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02). Monocytes were further isolated using human total monocyte isolation kit (Miltenyi Biotec, Cat: 130-096-537), and macrophage colony-stimulating factor (M-CSF, R&D Systems, Cat: 216-MC) was added. The wall was cultured for 7 consecutive days, and the mononuclear cells were cells induced to differentiate into macrophages. On the day of the phagocytosis test, the differentiated macrophages were starved in serum-free medium for 2 hours. At the same time, target tumor cells Raji were fluorescently labeled according to the labeling procedures recommended by the instructions of CFSE (eBioscience, Cat: 65-0850-85). Mix the labeled tumor cells and macrophages at a ratio of 4:1, and add the experimental antibody at the concentration to be tested and incubate at 37°C for 2 hours. The cells were then washed twice with PBS and digested with trypsin (Gibco, Cat: 25200072). APC-labeled anti-CD14 antibody (Biolegend, Cat: 325608) was added and placed on ice in PBS containing 2% fetal calf serum to protect from light. Incubate for 30 minutes. Cells were washed twice and analyzed by flow cytometry. Calculate the proportion of CFSE-positive cells in the CD14-positive macrophage population. As shown in Table 1, 125G4A4 can effectively promote the phagocytosis of tumor cells by macrophages.

Example 2 Humanization of fusion tumor antibodies

2.1 Determination of fusion tumor antibody variable region sequence

Using the fusion tumor sequencing method, the cells of the fusion tumor pure strain 125G4A4 were expanded and cultured, and total RNA was extracted using TRIzol (purchased from Ambio), and reverse transcribed into DNA using antibody-specific primers (Takara, PrimerScript 1 ^st Strand cDNA Synthesis Kit), and used antibody-specific primers to amplify the gene fragment encoding the mouse immunoglobulin V-region. Through sequence determination and analysis, the fusion tumor antibody variable region sequence, the heavy chain variable region of the 125G4A4 antibody, and The amino acid sequences of the light chain variable region are shown in SEQ.ID No.: 1 and 2 respectively, and the nucleotide sequences are shown in SEQ.ID No.: 19 and 20 respectively.

2.2 Construction and expression of chimeric antibodies

According to the mechanism of action of CD47, in specific embodiments of the present invention, the constant region of human IgG4 (S228P) is used as the heavy chain constant region of the antibody, and the human light chain kappa chain constant region is used as the antibody light chain constant region. Mutation of serine at position 228 of the IgG4 core hinge region to proline (S228P) can enhance the disulfide bonding of the core hinge region, reduce IgG4 Fab arm exchange, and greatly reduce the formation of half-molecules. After the heavy chain and light chain constant region genes are synthesized, the heavy chain and light chain variable region genes are homologously recombined into the vector through EcoRI and BamHI double enzyme digestion vector PTT5. After correct sequencing, the antibody heavy chain and light chain were co-transfected into HEK293 cells at a molar ratio of 1.5:1. After 120 hours of culture, the supernatant was collected by centrifugation and purified to obtain chimeric antibodies.

Before humanized antibody design, some post-translational modification (PTM) sites in the CDR region need to be mutated to avoid affecting the protein conformation and thus its function. After PTM analysis, two PTM sites were identified in the CDR of 125G4A4, including an NSS glycosylation site in the heavy chain and a DG isomerization site in the light chain. The NSS glycosylation site and DG isomerization site were mutated to QSS and EG, respectively. The mutated chimeric antibody purified according to this example was named Ch-125G4-m35. The amino acid sequences of the heavy chain variable region and light chain variable region of the Ch-125G4-m35 antibody are shown in SEQ. ID No.: 3 and 4 respectively.

2.3 Humanized design of chimeric antibodies

The chimeric antibody 125G4A4m antibody variable region sequence was Blast compared with the PDB_Antibody database, and the human skeleton sequence with the highest similarity was selected for humanization: 125G4A4 m heavy chain variable region and human germline IGHV1-69, and the light chain can The variable region has high sequence homology with human germline IGKV1-16. The Kabat assignment system is then used to define the amino acid sequence of the variable region CDR and its precise boundaries. Then, the CDR segment of the variable region of the mouse antibody is grafted into the human framework sequence to achieve humanization of the antibody.

In order to maintain the activity of the antibody, computer simulation technology can be used to analyze the variable region and its surrounding framework amino acid sequences using molecular docking to examine its three-dimensional binding mode. By calculating the electrostatic force, van der Waals force, hydrophobicity and entropy value, the key amino acid individuals in each candidate antibody gene sequence that can interact with CD47 and maintain the spatial structure are analyzed, and then grafted back to the selected human antibody gene framework. On this basis, the amino acid sites in the framework region that must be retained are marked to synthesize humanized antibodies. Some key sites in the antibody framework region need to be back mutated to the antibody framework region sequence of the chimeric antibody Ch-125G4-m35. Based on the number and arrangement of reverse mutations, multiple different humanized heavy chain variable regions (SEQ.ID No.: 5, SEQ.ID No.: 6, SEQ.ID No.: 7) and light chains were designed. Variable region (SEQ.ID No.: 8, SEQ.ID No.: 9, SEQ.ID No.: 10) (see Table 2). The humanized antibody Hu-125G4A4-48 finally identified in the present invention was named HMA02h14-48 in subsequent experiments. The amino acid sequences of the heavy chain variable region and light chain variable region of the antibody are shown in SEQ. ID No.: 7 and 8 respectively.

2.4 Expression of humanized antibodies

The DNA fragments of the humanized designed heavy chain and light chain variable regions were amplified and cloned into a vector containing the human antibody constant region to construct a plasmid expressing the antibody (pCDNA3.4, purchased from Thermo Cat# A14697) . The heavy chain and light chain expression vectors were co-transfected into Expire293 cells (Thermo Cat#A14525). After culturing at 37°C for 6 days, the supernatant was collected and purified by Protein A affinity according to the above method to obtain recombinant antibodies for antibody use. further characterization. Humanized antibodies are of the IgG4 S228P (IgG4P) subtype.

Example 3 Screening of humanized antibodies

Highly active humanized antibodies were screened by detecting the binding ability of humanized antibodies to cynomolgus monkey B cells, the ability of human macrophages to phagocytose tumor cells, and the agglutination ability of red blood cells.

Detection of the binding ability of humanized antibodies to monkey B cells: Flow cytometry was used to detect the binding of 125G4A4 humanized series antibodies to CD47 on the surface of cynomolgus monkey B cells. The specific method is as follows: from cynomolgus monkey blood (provided by Shanghai Yinuosi Biotechnology Co., Ltd.), Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02) was used to separate peripheral blood by density gradient centrifugation. Peripheral Blood Mononuclear Cells (PBMCs). PBMC were incubated with 125G4A4 humanized series antibodies or isotype (IgG4P) in PBS containing 2% fetal calf serum for 30 minutes at 4°C. The cells were then washed three times and incubated with secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2% fetal calf serum for 30 minutes at 4°C in the dark. Cells were washed three times and analyzed by flow cytometry. B cells were labeled with an anti-human CD20 antibody cross-reactive with cynomolgus monkey (Brilliant Violet 421 ^TM labeled anti-human CD20 Antibody, Biolegend, Cat: 302330), and flow cytometry was performed on Canto II (BD Biosciences). Detect and obtain its mean fluorescence intensity (MFI).

The ability of macrophages to phagocytose tumor cells and the agglutination ability of red blood cells were detected according to the methods described in Examples 1.5 and 1.4 respectively.

The results are shown in Table 5. The 125G4A4 humanized series of antibodies combined with CD47 expressed on cynomolgus monkey B cells under the test concentration conditions and promoted the phagocytosis of tumor cells Raji by macrophages. Among them, Hu-125G4A4m-48 was at 33nM. The phagocytosis efficiency is the strongest, and other activities are similar to those of the chimeric antibody Ch-125G4m-m35, with a smaller number of back mutations. Therefore, it was selected for further testing, and subsequent testing was named HMA02h14-48.

Example 4 FACS detection of the binding ability of HMA02h14-48 to tumor cells

Human Burkitt lymphoma cell line Raji cells (Shanghai Institute of Life Sciences, SIBS, CCL-86 ^TM /ATCC), human diffuse large cell lymphoma Toledo cells (ATCC ^® CRL-2631 ^TM )) and human mantle cell lymphoma REC -1 cells (ATCC ^® CRL-3004 ^TM ) endogenously express human CD47 on the surface. As described in the detection method described in Example 1.2, flow cytometry was used to detect the interaction between humanized antibody HMA02h14-48 and CD47 on the surface of the above tumor cells. combine. The highest antibody concentration was 667nM, with gradient dilution, and a total of 8 concentrations were tested.

The results are shown in Figure 1-3. Both HMA02h14-48 and Hu5F9 bind to CD47 expressed on the surface of tumor cells Raji, Toledo and REC-1 cells. The maximum fluorescence intensity of HMA02h14-48 when reaching the plateau phase is higher than that of Hu5F9. EC ₅₀ and maximum fluorescence intensity are shown in Table 6.

The negative isotype control antibody (isotype) used in this example and other examples is human IgG4P, purchased from Shanghai Ruizhi Chemical Research Co., Ltd.

Example 5 Biacore detection of affinity between antibody HMA02h14-48 and human CD47

Biacore determines binding kinetic parameters by measuring Surface Plasmon Resonance (SPR). This technology detects the microscopic rate constants of the binding (ka) and dissociation (kd) of antibodies and antigens, and calculates the affinity values between antibodies and antigens. Biacore instrument (Biacore T200) and reagents were purchased from GE Healthcare. Anti-human Fc antibody was immobilized on sensor chip CM5. The purified antibodies (HMA02H14-48 and Hu5F9) were diluted in mobile phase buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween-20, pH 7.4), and flowed through the CM5 chip plated with anti-human Fc antibodies. then The gradient-diluted human CD47-His (ACROBiosystems, Cat: CD7-H5227) fusion protein flows through the detection chip to measure the binding of the antigen and the antibody, and then the mobile phase buffer flows through the chip to detect the dissociation of the antigen and the antibody. Collect the binding and dissociation signal data of antigen and antibody at different concentrations, and calculate the affinity of antigen and antibody through 1:1 Langmuir model fitting.

The results are shown in Table 7. HMA02h14-48 binds to human CD47 with high affinity, and the K _D value is 7.77E-10(M).

Example 6 ELISA detection of the activity of HMA02h14-48 in blocking the interaction between human CD47 and SIRPα

As described in the aforementioned detection method in Example 1.3, ELISA was used to detect the ability of HMA02h14-48 to block the interaction between human CD47 and SIRPα. The highest antibody concentration was 67nM, with gradient dilution, and a total of 8 concentrations were tested.

The results are shown in Figure 4. The antibody HMA02h14-48 of the present invention blocks the interaction between human CD47 and SIRPα, with IC ₅₀ =1.58nM.

Example 7 Effect of HMA02h14-48 on the phagocytosis of tumor cells by human macrophages

According to the method described in Example 1.5, HMA02h14-48 was detected to promote the response of human macrophages to human Burkitt lymphoma cell line Raji cells, human diffuse large cell lymphoma Toledo cells, human mantle cell lymphoma REC-1 cells and human progeroblastic lymphoma cells. Myeloid leukemia cell line HL-60 Phagocytosis effect of cells. The highest antibody concentration was 100 μg/mL, with gradient dilution, and a total of 8 concentrations were tested.

The results show that compared with the control antibodies Hu5F9 and SRF231, HMA02h14-48 can promote the phagocytosis of human Burkitt lymphoma cell line Raji by macrophages, with the highest phagocytosis efficiency reaching 36.5%, at different concentrations of 0.1~100μg/ml. The phagocytosis rates were higher than those of Hu5F9 and SRF231. The highest phagocytosis efficiency of HMA02h14-48 for human mantle cell lymphoma REC-1 can reach 84.6%, and it can maintain the phagocytosis rate at about 70% at a low concentration of 0.1 μg/ml, which is higher than Hu5F9 and SRF231. HMA02h14-48 promotes the phagocytosis of Toledo cells by macrophages, and the phagocytosis rate can reach 94.2%. HMA02h14-48 can promote the phagocytosis of tumor cells HL-60 by macrophages, with the highest phagocytosis efficiency reaching 65%.

Example 8 Detection of the effect of HMA02h14-48 on the agglutination activity of red blood cells in vitro

Human red blood cells were diluted to 10% in PBS and incubated with instilled CD47 antibodies in a round-bottom 96-well plate at room temperature for 16 hours. The presence of unprecipitated red blood cells is evidence of blood cell aggregation. Compared with unaggregated red blood cells that precipitate to form white dots, the unprecipitated red blood cells appear in a reticular shape with an area larger than the negative isotype control antibody (see Figure 9). The test results of negative isotype control antibodies (Isotype) were used as normal standards.

The antibody HMA0214-48 was tested for promoting erythrocyte agglutination according to the method described in the aforementioned Example 1.4. The highest antibody concentration was 667nM, with gradient dilution, and a total of 12 concentrations were tested.

The results are shown in Figure 9. The CD47 antibody Hu5F9 shows that red blood cells will significantly agglutinate at a concentration equal to or higher than 0.9nM, while the antibody HMA02h14-48 of the present invention has no obvious agglutination of human red blood cells in vitro at different concentrations of 0.004 to 667nM. of agglutination.

Example 9 FACS detection of the binding ability of HMA02h14-48 to human red blood cells

It is known in the prior art that when therapeutic anti-CD47 antibodies are used clinically, the side effect of anemia often occurs. It is generally believed that anti-CD47 antibodies bind to CD47 on the surface of red blood cells, thereby causing macrophages to engulf red blood cells, which may be another major cause of anemia. In the present invention, we use flow cytometry to detect the binding ability of HMA02h14-48 to human red blood cells to assess the risk of antibodies. Specifically, red blood cells derived from healthy donors were incubated with diluted HMA02h14-48 (the highest concentration was 667 nM, a total of 8 tested concentrations) in PBS containing 2% fetal calf serum for 30 minutes at 4°C. The cells were then washed three times and incubated with secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2% fetal calf serum for 30 minutes at 4°C in the dark. The cells were washed three times with PBS containing 2% fetal bovine serum (FBS), and the fluorescence signal was detected by a Canto II (BD Biosciences) flow cytometer, and based on its mean fluorescence intensity (MFI), GraphPad was used to Fit the concentration-dependent curves and calculate the _EC50 .

The results are shown in Figure 10. The maximum average fluorescence intensity of HMA02h14-48 binding to CD47 on the surface of human red blood cells is lower than that of the control antibody Hu5F9. Its EC ₅₀ maximum average fluorescence intensity is shown in Table 9.

Example 10 Inhibition of Toledo tumor growth by humanized antibody HMA02h14-48

Purpose: To establish a Toledo subcutaneous tumor model on NOD-Scid mice and study the anti-tumor activity of the antibody of the present invention.

Methods: Human diffuse large cell lymphoma cells Toledo (ATCC® CRL-2631 ^TM ) were cultured in RPMI1640 medium containing 10% fetal calf serum. Tumor cells were suspended in RPMI1640 and implanted subcutaneously into the right flank of male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd.) at a dose of 1×10 ⁷ cells/mouse.

Fifteen days after tumor cell inoculation, the mice were randomly divided into 6 groups according to tumor volume. Hu5F9 and HMA02h14-48 antibodies were diluted in PBS and administered at a dose of 10 mg/kg according to the schedule shown in Table 10. Negative isotype control antibody (isotype) IgG4P was purchased from Shanghai Ruizhi Chemical Research Co., Ltd.

Tumor volume (tumor volume = 0.5 × long diameter × short diameter 2) and mouse body weight were measured regularly. Student t test in Excel software was used to perform statistical analysis on tumor volume changes and body weight. p <0.05 was considered a significant statistical difference. The tumor regression rate of each antibody treatment group after administration was statistically calculated.

The calculation formula for the tumor regression rate in each treatment group is: [(D ₀ average tumor volume - _{D t} average tumor volume)/D ₀ average tumor volume] × 100%.

The relative weight calculation formula of mice is: (mouse weight on the day of measurement/mouse weight at the time of grouping) × 100%.

result:

The experimental results are shown in Table 11 and Figure 11.

The tumors in the Isotype control antibody group grew well, while in the other treatment groups, after administration of the antibodies, the subcutaneous tumors gradually shrank in size from the initial size until they completely disappeared. Among them, each dose group of Hu5F9 and HMA02h14-48 antibodies achieved complete tumor disappearance (regression rate 100%) when measured on the 11th day. Compared with the control antibody control group, the tumor volume changes were extremely significant. And after stopping the drug, the animals were continuously observed until the 67th day, and there was still no sign of tumor re-growth. In addition, the animals in each dose group of HMA02h14-48 were in good condition, and there was no significant difference in the weight of mice on the 21st day compared with before treatment. The body weight of the Hu5F9 high-dose group on day 21 was about 5% lower than that on day 0, but there was no statistical difference compared with the starting weight ( p >0.05); while there was no weight loss in the low-dose group, suggesting that The effect of Hu5F9 on body weight has a certain dose-effect relationship.

Based on the above data, both Hu5F9 and HMA02h14-48 antibody treatments have extremely significant anti-tumor effects. A single dose of 10 mg/kg can completely eliminate tumors and last for a long time.

Example 11 Inhibition of REC-1 tumor growth by humanized antibody HMA02h14-48

Purpose: To establish a REC-1 subcutaneous tumor model on NOD-Scid mice and study the anti-tumor activity of the antibody of the present invention.

Methods: Human mantle cell lymphoma cells REC-1 (ATCC ^® CRL-3004 ^TM ) were cultured in RPMI1640 medium containing 10% fetal calf serum. Tumor cells were suspended in RPMI1640 and implanted subcutaneously into the right flank of male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd.) at a dose of 5×10 ⁶ cells/mouse.

Eleven days after tumor cell inoculation, the mice were randomly divided into 5 groups according to tumor volume. Hu5F9 and HMA02h14-48 antibodies were diluted with PBS and administered according to the schedule shown in Table 12. Antibody Hu5F9 was prepared by GenScript, and antibody HMA02h14-48 was prepared according to the method of Example 2. Isotype control antibody (isotype) IgG4p was purchased from Shanghai Ruizhi Chemical Research Co., Ltd.

Tumor volume (tumor volume = 0.5 × long diameter × short diameter ² ) and mouse body weight were measured regularly. The tumor inhibition rate and regression rate of the antibody treatment group on the 12th day after administration were calculated.

The calculation formula for the tumor inhibition rate is: [(average tumor volume change in the control group - average tumor volume change in the administration group)/average tumor volume change in the control group] × 100%. Student t test in Excel software was used to perform statistical analysis on tumor volume changes and body weight. p<0.05 was considered a significant statistical difference.

The calculation formula for the tumor regression rate in each treatment group is: [(D0 average tumor volume - Dt average tumor volume)/D0 average tumor volume] × 100%.

The relative weight calculation formula of mice is: (mouse weight on the day of measurement/mouse weight at the time of grouping) × 100%.

result:

The experimental results are shown in Table 13 and Figure 12.

Twelve days after administration, compared with the Isotype group, antibody Hu5F9 inhibited tumor growth by 16.7% at a single dose of 3 mg/kg (p>0.05); antibody HMA02h14-48 inhibited tumor growth at a single dose of 1 mg/kg. The inhibition rates of tumor growth at doses of kg, 3mg/kg and 10mg/kg were 3.8% (p>0.05), 54.7% (p<0.01) and 107.2% (p<0.001) respectively. Among them, the HMA02h14-48 antibody high-dose group achieved complete tumor disappearance (regression rate 100%) when measured on the 10th day. In addition, there was no significant difference in the relative body weight of mice in each antibody treatment group.

Based on the above data, in the REC-1 model, HMA02h14-48 antibody treatment is dose-dependent, and a single administration of 10 mg/kg can completely eliminate the tumor.

Materials and methods

在製劑的製備過程中使用的主要材料信息。

Information on the main materials used in the preparation of the dosage form.

製劑穩定性檢測項目和檢測方法

Preparation stability testing items and testing methods

Protein content (unit: mg/mL)

The protein concentration in the sample was determined using a UV spectrophotometer (LUNATIC, Lunatic-16).

Size exclusion chromatography (SEC-HPLC)

This method mainly separates molecules based on their size or hydrodynamic radius differences. By SEC-HPLC, antibodies can be separated into three main forms: high molecular weight forms (High Molecular Weight Species, HMMS), main peaks (mainly antibody monomers), and low molecular weight forms (Low Molecular Weight Species, LMMS). Antibody purity can be calculated as the percentage of the main peak area in the chromatogram to the sum of all peak areas. By SEC-HPLC method, the percentage of antibody monomers in the preparation product, as well as the content of soluble aggregates and fragments can be measured. For further description of the SEC-HPLC method, see, for example, 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).

The following parameters can be used for SEC-HPLC detection:

Chromatography column: TSK-gel ^® G3000SWXL (7.8×300mm, 5μm)

Mobile phase: 100mM PB, 100mM Na ₂ SO ₄ ‧10H ₂ O, pH 6.7±0.1

Flow rate: 0.7mL/min

Column temperature: Not controlled

Detection wavelength: 280nm

Loading volume: 1μL

Sampler temperature: 15℃

Running time: 25min

Loading concentration: 50mg/mL

Non-reducing capillary gel electrophoresis (CE-SDS-NR)

The non-reducing CE-SDS method is a method for determining the purity of monoclonal antibodies using a capillary tube as a separation channel. In CE-SDS, protein migration is driven by surface charge induced by SDS binding, which is proportional to the protein's molecular weight. 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 a capillary molecular sieve gel matrix. This method has been widely used to monitor the purity of denatured intact antibodies. For further description of the CE-SDS method, see, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

Briefly, add 100 μg protein into a centrifuge tube, add sample buffer sodium dodecyl sulfate solution (SDS-MW) to a final volume of 95 μL, then add 2 μL internal standard (10kDa protein), and 5 μL 250mM iodoacetamide. (IAM), mix and incubate at 70°C for 10 minutes. After incubation, cool the sample to room temperature and transfer 90 μL to a PCR tube. CE-SDS separation was performed on (SCIEX production, model PA800 PLUS). Use a UV detector to detect protein migration and obtain an electrophoresis spectrum. The purity of an antibody preparation can be calculated as the percentage of the peak area of the main IgG peak to the sum of all peak areas.

Imaging capillary isoelectric focusing electrophoresis (icIEF method)

This method can provide a quantitative distribution of charge variants. icIEF achieves the purpose of molecular separation based on the charge difference (apparent pI value) of molecules in the pH gradient. In icIEF, the separation column is usually a short capillary (e.g., 5 cm long, 100 μm inner diameter silica capillary). White matter is focused in a capillary column at high voltage, and focus is monitored online in real time by a full-column imaging detection system operating at 280nM. One advantage of this technology is that various charge variants of the antibody sample can be recorded simultaneously with this full-column detection system. Generally, in icIEF, the sample is mixed with urea and icIEF buffer containing methylcellulose, pi molecular weight standards, and ampholytes. Then, you can use an icIEF column such as the icIEF column assembled by Protion Simple on an icIEF analyzer such as the iCE280 analyzer (Protein Simple, Santa Clara, CA). After the sample is focused for a certain period of time, the absorbance at 280 nm is measured to obtain the focused mAb. Spectra of charge variants. In the iCEIF spectrum, the protein-related peaks eluted before the main peak (i.e., the main component) are classified as acidic components; conversely, the protein-related peaks eluted after the main peak are classified as basic components. The relative amounts of the main components, acidic components and basic components 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 interlaboratory study, J Sep Sci. 2012 Nov; 35(22): 3124-9.doi: 10.1002 /jssc.201200633.Epub 2012 Oct 15.

Briefly, icIEF detection can be performed as follows: dilute (or desalt) the antibody sample to approximately 5mg/mL, take 4μL into 96μL buffer (the buffer contains 4% ampholytes, 0.35% methylcellulose, 1% PI marker (propidium iodide marker), 500mM arginine, 4M urea). After mixing, the imaging capillary isoelectric focusing spectrum was analyzed on a Maurice protein characterization analyzer (Protein Simple, Santa Clara, CA).

Protein activity (ELISA)

ELISA (enzyme-linked immunoassay test) can be used to detect the binding ability of the antibody in the preparation to its antigen and evaluate the biological activity of the antibody.

Osmotic pressure

The osmolality of the formulation samples was measured on a 3250 osmometer from Advanced Instruments based on the freezing point depression principle.

pH

The pH value of the preparation sample was measured by potentiometric method using a glass electrode. The instrument used was METTLER TOLEDO's S400-B pH meter. Before measuring the sample, the pH meter was calibrated with standard solutions with pH values of 4.01, 7.00 and 9.21.

Example 12. Pharmaceutical preparation stability study 1

The antibody HMA02h14-48 prepared in Example 2 above was used in the example of pharmaceutical preparation. This antibody was called mAbl in pharmaceutical preparation research.

Design each group of candidate formulations as shown in Table 16, and examine the effects of different pH/buffers, antibody concentrations, stabilizer or surfactant concentrations on antibody stability.

Experimental methods

1) Take mAb1 antibody samples respectively (sample information is shown in Table 15, in which the citrate buffer system is citric acid-sodium citrate buffer), and add the corresponding buffer solution and arginine hydrochloride stock solution according to the scheme in Table 16 , adjust arginine hydrochloride to the target concentration. For E6, add citrate buffer mother solution at the same time to adjust citrate to the target concentration; for E3 and E5, use histamine buffer corresponding to arginine hydrochloride. Change the liquid and concentrate the antibody, and concentrate other prescription samples directly; 2) Add the corresponding sucrose mother solution (when needed) and polysorbate 80 mother solution; 3) Add the corresponding buffer solution to adjust each component to the target concentration; 4) Filter and sterilize with a 0.22μm disposable sterile filter in a biological safety cabinet, aliquot (1mL/bottle), add stoppers, cap and label.

Investigation plan: According to the sampling plan in Table 17, conduct stability investigation at 40°C high temperature and freeze-thaw (-70°C±10°C/5°C±3°C).

In Table 16, the citrate buffer is citric acid-sodium citrate buffer, and the histamine buffer is histidine acid-histidine hydrochloride buffer.

X：蛋白含量，SEC-HPLC,CE-SDS-NR,icIEF；Y：ELISA

X: protein content, SEC-HPLC, CE-SDS-NR, icIEF; Y: ELISA

Results and analysis

(1) Protein content test results

The protein content detection results are shown in Table 18. At T0, the protein content of each candidate prescription sample met the experimental design requirements and there was no significant difference. Compared with T0, after 5 cycles of freezing and thawing at -70°C/5°C and 4 weeks of high temperature storage at 40°C, the protein content of each candidate formulation showed no significant change, and the high temperature and freeze-thaw stability was good.

(2)SEC-HPLC purity

The SEC-HPLC purity test results are shown in Tables 19 and 20. At T0, there was no significant difference in the SEC-HPLC purity of each candidate prescription sample.

Compared with T0, with the extension of the high-temperature investigation time at 40°C, the purity of the SEC-HPLC main peak of all recipes did not change significantly, and the high-temperature stability was good.

The freeze-thaw inspection results showed that compared with T0, after 5 cycles of freeze-thaw, the SEC-HPLC purity of each prescription sample did not change significantly, indicating that the mAb1 antibodies in each candidate prescription have good freeze-thaw stability.

(3)CE-SDS-NR purity

The CE-SDS-NR purity test results are shown in Table 21. The research results showed that at T0, there was no significant difference in the purity of CE-SDS-NR among the candidate formulations. Compared with T0, after being placed at 40°C for 4 weeks, the monomer purity of all candidate prescription samples did not change significantly, and the high temperature stability was good.

After 5 freeze-thaw cycles, the monomer purity of all candidate prescriptions did not change significantly, and there was no significant difference between the prescriptions.

(4)icIEF purity

The icIEF purity test results are shown in Tables 22 and 23. At T0, there was no significant difference in the icIEF results of each candidate prescription sample. Compared with T0, after 4 weeks of investigation at 40°C, the purity of the main peak of each prescription did not change significantly, and the high temperature stability was good.

After 5 cycles of freezing and thawing conditions, there was no significant change in the main peak purity, acid peak and alkali peak content of all prescriptions, and the freeze-thaw stability was good.

(5) ELISA method to detect relative protein binding activity

The activity test results are shown in Table 24. At T0, the activity of each candidate prescription sample was in the range of 70% to 130%, and there was no significant difference. Compared with T0, after being placed at 40℃ for 4 weeks And after 5 freeze-thaw cycles, the activity of each prescription was maintained in the range of 70% to 130%, and all met the quality requirements.

In summary, no significant differences were found in protein content, SEC-HPLC, CE-SDS-NR and icIEF purity between different pharmaceutical preparations within 4 weeks of high temperature testing at 40°C or after 5 cycles of freezing and thawing conditions. It has high temperature stability and good freeze-thaw stability.

Example 13: Pharmaceutical preparation stability study 2

Based on the aforementioned research results, under isotonic conditions, the pharmaceutical formulation was designed to further compare and confirm 1) the impact of the ratio of arginine hydrochloride and sucrose, and 2) the impact of polysorbate 80 concentration. To understand the stability performance of the preparation at the marginal dosage of arginine hydrochloride, sucrose, and polysorbate 80 in the prescription.

Experimental methods

Take mAb1 antibody samples respectively (see Table 25 for sample information, in which the citrate buffer system is citric acid-sodium citrate buffer), and add the corresponding buffer solution (20mM citrate buffer) according to the protocol in Table 26 (It is citric acid-sodium citrate buffer), 1) For F1, F2, F7, and F8, add arginine hydrochloride mother liquor and polysorbate 80 mother liquor, and finally add buffer to adjust each component to the target Concentration; 2) For F3 and F4, add arginine hydrochloride mother liquor, sucrose mother liquor and polysorbate 80 mother liquor, and add buffer to adjust each component to the target concentration; 3) For F5, add the corresponding sucrose mother liquor and polysorbate 80 mother liquor. Sorbitate 80 stock solution, and finally add buffer to adjust each component to the target concentration; 4) For F6, use 20mM citrate buffer to dilute arginine hydrochloride to the target concentration, then concentrate the antibody, and then add the corresponding Sucrose stock solution and polysorbate 80 stock solution, and add buffer to adjust each component to the target concentration; 5) Filter and sterilize with a 0.22 μm disposable sterile filter in a biological safety cabinet, aliquot (1 mL/bottle), stopper, and seal Cover and label.

Investigation plan: Conduct a 40°C high temperature stability investigation according to the sampling plan in Table 27.

X：蛋白含量，SEC-HPLC,CE-SDS-NR,icIEF；Y：ELISA

X: protein content, SEC-HPLC, CE-SDS-NR, icIEF; Y: ELISA

Experimental results

(1) Osmotic pressure test results

The osmotic pressure test results of each candidate drug preparation are shown in Table 28. The osmotic pressure of all candidate prescription samples is isotonic.

(2) Protein content test results

The protein content detection results are shown in Table 29. At T0, the protein concentration of each candidate prescription sample meets the experimental design requirements, and there is no significant difference between the prescriptions. Compared with T0, after being placed at 40°C for 4 weeks, the protein content of each candidate formulation showed no significant change, and the stability at 40°C was good.

(3)SEC-HPLC purity

The SEC-HPLC purity test results are shown in Table 30. At T0, there was no significant difference in the SEC-HPLC purity of each candidate prescription sample. Compared with T0, after 4 weeks of investigation at high temperature of 40°C, the purity of the SEC main peak of all recipes did not change significantly, and the high temperature stability of 40°C was good.

(4)CE-SDS-NR purity

The CE-SDS-NR purity test results are shown in Table 31. The research results showed that at T0, there was no significant difference in the purity of CE-SDS-NR among the candidate formulations. Compared with T0, after being placed at high temperature of 40°C for 4 weeks, the monomer purity of all candidate prescription samples did not change significantly, and the high temperature stability of 40°C was good.

(5)icIEF purity

The icIEF purity test results are shown in Table 32. Compared with T0, after 4 weeks of investigation at high temperature of 40°C, the purity of the main peak of each prescription did not change significantly, and the stability at high temperature of 40°C was good.

(6) ELISA method to detect relative protein binding activity

The activity test results are shown in Table 33. At T0, the activity of each candidate prescription sample was in the range of 70% to 130%, and there was no significant difference. Compared with T0, after being placed at high temperature 40°C for 4 weeks, the activity of each prescription was maintained in the range of 70% to 130%, showing good activity stability and no obvious difference between the prescriptions.

In summary, after 4 weeks of high-temperature investigation at 40°C, there was no significant difference in protein content, SEC-HPLC, CE-SDS-NR and icIEF purity between different formulations. It was not found that different concentration ratios of arginine hydrochloride and sucrose had a significant impact on protein stability.

Sequence of the invention:

The exemplary embodiments and examples of the present invention have been described above. It should be understood by those of ordinary skill in the art that these disclosures are only exemplary, and various other substitutions, adaptations and modifications can be made within the scope of the present invention. . Therefore, the present invention is not limited to the specific embodiments and examples set forth herein.

TW202330027A_111138267_SEQL.xml

Claims (36)

A stable pharmaceutical preparation containing (i) anti-CD47 antibody or antigen-binding fragment thereof; (ii) Buffer; (iii) stabilizers; and (iv) Surfactant; pH value is about 5.0 to about 8.5, and wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises (1) One to three selected from HCDR1, HCDR2 and HCDR3 of the heavy chain variable region (VH), wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 6 or 7; and / or (2) One to three selected from LCDR1, LCDR2 and LCDR3 of the light chain variable region (VL), wherein the VL contains the amino acid sequence shown in SEQ ID NO: 2, 4, 8, 9 or 10 . The pharmaceutical preparation according to claim 1, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 of VH, and LCDR1, LCDR2 and LCDR3 of VL; wherein said VH and VL are selected from: (1) VH comprising the amino acid sequence shown in SEQ ID NO: 1 and VL comprising the amino acid sequence shown in SEQ ID NO: 2; (2) VH comprising the amino acid sequence shown in SEQ ID NO: 3 and VL comprising the amino acid sequence shown in SEQ ID NO: 4; (3) VH comprising the amino acid sequence shown in SEQ ID NO: 5 and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9 or 10; (4) VH comprising the amino acid sequence shown in SEQ ID NO: 6 and VL comprising the amino acid sequence shown in SEQ ID NO: 9 or 10; or (5) VH containing the amino acid sequence represented by SEQ ID NO:7 and VL containing the amino acid sequence represented by SEQ ID NO:8, 9 or 10. The pharmaceutical preparation according to claim 1, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises (1) Selected from one to three of heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 12 The amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 13 or 17 or 21; and/or (2) One to three selected from the group consisting of light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 14, the LCDR2 comprising SEQ ID NO: 15 or The amino acid sequence shown in SEQ ID NO: 16, the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16. The pharmaceutical preparation according to claim 3, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises (1) Heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, wherein the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 12 and The HCDR3 includes the amino acid sequence shown in SEQ ID NO: 13 or 17 or 21; and/or (2) Light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, wherein the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 14, and the LCDR2 includes the amine shown in SEQ ID NO: 15 or 18 or 22 The amino acid sequence and the LCDR3 include the amino acid sequence shown in SEQ ID NO: 16. The pharmaceutical preparation according to claim 3, wherein the anti-CD47 antibody or antigen-binding fragment thereof (1) The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 12, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 17 sequence; and/or (2) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 14, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 18, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 16 sequence. The pharmaceutical preparation according to any one of claims 1 to 3, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises (1) Heavy chain variable region (VH), which contains an amino acid sequence identical to or at least 90%, 91%, 92%, 93% identical to the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 6 or 7 , an amino acid sequence with 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and/or (2) Light chain variable region (VL), which contains an amino acid sequence identical to or at least 90%, 91%, 92%, 93% identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 8, 9 or 10 , an amino acid sequence with 94%, 95%, 96%, 97%, 98% or 99% sequence identity. The pharmaceutical preparation according to claim 6, wherein the VH includes the amino acid sequence shown in any one of SEQ ID NO: 1, 3, 5, 6 or 7, and the VL includes SEQ ID NO: 2, 4, 8 The amino acid sequence shown in any one of , 9 or 10. The pharmaceutical preparation as claimed in claim 7, wherein the VH and VL are selected from: (1) VH comprising the amino acid sequence shown in SEQ ID NO: 1; and VL comprising the amino acid sequence shown in SEQ ID NO: 2; (2) VH comprising the amino acid sequence shown in SEQ ID NO: 3; and VL comprising the amino acid sequence shown in SEQ ID NO: 4; (3) VH comprising the amino acid sequence shown in SEQ ID NO: 5; and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9 or 10; (4) VH comprising the amino acid sequence shown in SEQ ID NO: 6; and VL comprising the amino acid sequence shown in SEQ ID NO: 9 or 10; or (5) VH comprising the amino acid sequence represented by SEQ ID NO: 7; and VL comprising the amino acid sequence represented by SEQ ID NO: 8, 9 or 10. The pharmaceutical preparation of claim 1, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 7 The amino acid sequence shown in SEQ ID NO: 8, the VL contains the amino acid sequence shown in SEQ ID NO: 8. The pharmaceutical preparation according to any one of claims 1 to 9, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain constant region, such as a human IgG1 constant region, a human IgG1TM constant region, a human IgG4 constant region or a human IgG4P constant region. The pharmaceutical preparation according to any one of claims 1 to 10, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises a light chain constant region, such as a human light chain kappa chain constant region. The pharmaceutical preparation according to any one of claims 1 to 11, wherein the anti-CD47 antibody is a murine antibody, a chimeric antibody or a humanized antibody. The pharmaceutical preparation according to any one of claims 1 to 12, wherein the anti-CD47 antibody or antigen-binding fragment thereof is a full-length antibody, a single-chain antibody, a single-domain antibody such as VHH, Fab, Fab', Fab'-SH , (Fab') ₂ , single chain antibody such as scFv, Fv, dAb (domain antibody) or bi(poly)specific antibody. The pharmaceutical preparation according to any one of claims 1 to 13, wherein the buffer is a histamine buffer or a citrate buffer. The pharmaceutical preparation according to any one of claims 1 to 14, wherein the concentration of the buffer is about 10mM to about 80mM, preferably about 20mM to about 50mM, for example, about 20mM or about 50mM. The pharmaceutical preparation according to any one of claims 1 to 15, wherein the stabilizer is selected from at least one of sugars, amino acids and polyols. The pharmaceutical preparation according to any one of claims 1 to 16, wherein the stabilizer contains at least one amino acid, such as arginine, preferably arginine hydrochloride. The pharmaceutical preparation according to any one of claims 1 to 17, wherein the stabilizer consists of amino acids and sugar, preferably arginine hydrochloride and sucrose. The pharmaceutical preparation according to any one of claims 16 to 18, wherein the concentration of the amino acid is about 1 mg/mL to about 100 mg/mL, such as 5 mg/mL to 40 mg/mL. The pharmaceutical preparation according to any one of claims 16 to 19, wherein the concentration of the sugar is about 10 mg/mL to about 100 mg/mL, preferably about 20 mg/mL to about 80 mg/mL. The pharmaceutical preparation according to any one of claims 16 to 20, wherein the mass concentration ratio of the amino acid and sugar is about 1:1 to about 1:15. The pharmaceutical preparation according to any one of claims 1 to 21, wherein the surfactant is selected from polysorbate surfactants, preferably polysorbate-80 or polysorbate-20. The pharmaceutical preparation according to any one of claims 1 to 22, wherein the concentration of the surfactant is about 0.002% (w/v) to about 0.5% (w/v), preferably about 0.01% ( w/v) to about 0.05% (w/v), for example, about 0.01% (w/v), about 0.02% (w/v) or about 0.03% (w/v), preferably about 0.02% (w/v). The pharmaceutical preparation according to any one of claims 1 to 23, wherein the concentration of the anti-CD47 antibody is about 10-150 mg/mL, preferably about 40-120 mg/mL, for example, about 20 mg/mL, About 50 mg/mL, about 60 mg/mL, about 80 mg/mL, about 100 mg/mL, or about 110 mg/mL. The pharmaceutical preparation according to any one of claims 1 to 24, wherein the pH value is from about 5.5 to about 7.0, preferably from about 6.0 to about 7.0. The pharmaceutical preparation according to any one of claims 1 to 13, which contains: (i) About 10-150 mg/mL of anti-CD47 antibody or antigen-binding fragment thereof; (ii) about 10-80mM citrate buffer or histamine buffer; (iii) about 5-40 mg/mL arginine hydrochloride and about 0-80 mg/mL sucrose; and (iv) from about 0.01% (w/v) to about 0.05% (w/v) polysorbate-80; The pH value is about 6.0-7.0. The pharmaceutical preparation according to any one of claims 1 to 13, which contains: (i) About 40-120 mg/mL of anti-CD47 antibody or antigen-binding fragment thereof; (ii) about 20-50mM citrate buffer or histamine buffer; (iii) about 5-40 mg/mL arginine hydrochloride and about 20-80 mg/mL sucrose, wherein the mass concentration ratio of arginine hydrochloride and sucrose is about 1:1 to about 1:15, (iv) from about 0.01% (w/v) to about 0.05% (w/v) polysorbate-80; The pH value is about 6.0-7.0. The pharmaceutical preparation according to any one of claims 1 to 13, which contains: (i) About 40-120 mg/mL of anti-CD47 antibody or antigen-binding fragment thereof; (ii) about 20-50mM citrate buffer; (iii) about 5-40 mg/mL arginine hydrochloride; and (iv) from about 0.01% (w/v) to about 0.05% (w/v) polysorbate-80; The pH value is about 6.0-7.0. The pharmaceutical preparation according to any one of claims 1 to 28, which is in liquid form and further contains a solvent, such as water. A pharmaceutical preparation in freeze-dried form, which is prepared by freeze-drying the pharmaceutical preparation as described in any one of claims 1 to 29. A reconstituted pharmaceutical preparation, which is prepared by reconstituting the freeze-dried pharmaceutical preparation as described in claim 30 with a reconstitution medium, the reconstitution medium being selected from the group consisting of water for injection, bacteriostatic water for injection, sodium chloride solution and at least one of the glucose solutions. The reconstituted pharmaceutical preparation according to claim 31, further comprising a solvent, such as water. Use of a pharmaceutical preparation as described in any one of claims 1 to 29, a pharmaceutical preparation in freeze-dried form as claimed in claim 30, and/or a reconstituted pharmaceutical preparation as described in claim 31 or 32 , which is used to prepare a medicament for treating a CD47-related disease in a subject in need thereof, wherein the CD47-related disease is preferably cancer, and the cancer includes blood cancer and solid tumors, such as bladder cancer, pancreatic cancer, lymphoma, Leukemia, multiple myeloma, melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, endometrial cancer, kidney cancer, birthmark tumor, prostate cancer, thyroid cancer, cervical cancer, Gastric cancer, liver cancer, colon cancer, ovarian cancer, urothelial cancer, etc. An article of manufacture comprising a pharmaceutical preparation as described in any one of claims 1 to 29, a pharmaceutical preparation in freeze-dried form as claimed in claim 30, and/or a reconstitution as claimed in claim 31 or 32 Containers for pharmaceutical preparations. A method of treating or preventing CD47-related diseases in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical preparation as described in any one of claims 1 to 29, as described in claim 30 A pharmaceutical preparation in lyophilized form, and/or a reconstituted pharmaceutical preparation as described in claim 31 or 32. The method of claim 35, wherein the CD47-related disease is preferably cancer, including blood cancer and solid tumors, such as bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, melanoma, Leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, endometrial cancer, kidney cancer, birthmark tumor, prostate cancer, thyroid cancer, cervical cancer, stomach cancer, liver cancer, colon cancer, ovarian cancer, Urothelial carcinoma, etc.

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