Pharmaceutical composition of recombinant humanized anti-CD73 monoclonal antibody

Abstract

Provided is a pharmaceutical composition comprising (i) an anti-CD73 antibody, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant. Also provided is the use of the pharmaceutical composition.

Description

Pharmaceutical composition of recombinant humanized anti-CD73 monoclonal antibody

[0001] Citation of Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202310907284.1 filed on July 24, 2023, the entire contents of which are incorporated herein by reference in their entirety and for all purposes. Technical Field

[0003] The present application relates to a recombinant humanized anti-CD73 monoclonal antibody pharmaceutical composition preparation, a preparation method of the preparation, and uses thereof. Background Art

[0004] CD73 is an ecto-5'-nucleotidase encoded by the NT5E gene. It consists of two parts, the N-terminus and the C-terminus, and is anchored to the outer surface of the cell membrane through glycosylphosphatidylinositol (GPI) to form a homodimer. CD73 is widely expressed on the surface of human endothelial cells, lymphocytes, and other cells. It is highly expressed in various tumor tissues, including gastric cancer, renal cancer, prostate cancer, breast cancer (such as triple-negative breast cancer), and non-small cell adenocarcinoma, and is associated with a poor prognosis. CD73 can hydrolyze extracellular adenosine monophosphate (AMP) to adenosine. Adenosine is a powerful immunosuppressive molecule that weakens anti-tumor immunity by inhibiting the function of protective immune cells (such as effector T cells, NK cells, DCs, and B cells) while maintaining the function of regulatory immune cells (such as Tregs, MDSCs, TAMs, and CAFs), thereby participating in tumor immune escape. In addition to inhibiting immune cell function, a growing number of studies have shown that CD73 can also directly stimulate tumor cell proliferation, migration, invasion, and tumor angiogenesis. Inhibiting the ecto-nucleotidase CD73 can reverse adenosine-mediated immunosuppression. The study further found that hypoxia and chemoradiotherapy lead to upregulation of PDL1, CD73, and CD47 expression. Overall, overexpression of CD73 in tumors not only leads to metastasis and anthracycline resistance, but also causes immune escape due to dysregulated adenosine production. For these reasons, CD73 inhibitors are now combined with existing cancer treatments, including anti-PD-1 / anti-PD-L1 therapy, for cancer immunotherapy. Therefore, therapeutic strategies targeting CD73 have the potential to be applied clinically as monotherapy or combination therapy. In terms of research progress, there are currently nearly 30 anti-CD73 antibody products under development, including Medimmune's oleclumab and Tianjin Biopharma's uliledlimab. The most advanced anti-CD73 antibody is in Phase III clinical trials (oleclumab), and there are currently no marketed products. Therefore, the development and application of CD73 antibodies is urgently needed in this field, and the existing technology has a far-reaching unmet therapeutic need for anti-CD73 antibody-related products.

[0005] In order to treat the needs, the medicine (particularly antibody drug etc.) that generally should be given with high dose or multiple dose is given via intravenous injection usually, and this injection needs about 90 minutes or longer time, intravenous injection also follows other preparation procedure, is therefore all inconvenient for patient, doctor and medical staff, and can produce other cost.By contrast, subcutaneous injection has the advantage that can give immediately, but compared with intravenous injection, absorption rate is relatively low, and when injection volume is 3-5mL or more time, it may cause swelling and pain at the injection site because of absorbing slow generation.For this reason, subcutaneous injection protein therapeutic agent is limited to a small amount of 2mL or solution injection still less usually.Yet, after hyaluronidase is subcutaneously given (or subcutaneous injection) together with therapeutic drug, the hyaluronic acid that is distributed in the extracellular matrix is ​​hydrolyzed under the effect of hyaluronidase, thereby the viscosity of subcutaneous area reduces and the permeability of material increases, so the medicine of high dose or multiple dose can be easily delivered in body.

[0006] In addition, due to the existence of biological, chemical and physical barriers, as well as the strict requirements on long-term storage, osmotic pressure, solubility and stability of antibody drug preparations, the delivery of antibody bioactive agents to mammals by injection has become an issue that requires intensive research. Therefore, improved injectable preparations of antibodies are needed.

[0007] SUMMARY OF THE INVENTION

[0008] In one aspect, the present application provides a pharmaceutical composition comprising (i) an anti-CD73 antibody or an antigen-binding portion or functional fragment thereof, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.

[0009] On the other hand, the present application provides the use of the pharmaceutical composition in the preparation of a medicament for treating a tumor in an individual, such as a tumor that highly expresses CD73.

[0010] In another aspect, the present application provides a method for treating a tumor in an individual, such as a tumor that highly expresses CD73, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG1A shows the results of volume changes of transplanted tumors in CT26 tumor-bearing mouse groups resulting from administration of the exemplary humanized antibody Hab1F2F9.15 and other test substances.

[0013] FIG1B shows the results of changes in the volume of individual transplanted tumors in each group of CT26 tumor-bearing mice after administration of the exemplary humanized antibody Hab1F2F9.15 and other test substances.

[0014] Figure 2 shows the Tonset residual plot and main effect plot for the first round of excipient screening.

[0015] Figure 3 shows the CEX-HPLC residuals and main effects plots for the first round of excipient screening.

[0016] Figure 4 shows the Tonset residual plot and main effect plot for the second round of excipient screening.

[0017] Figure 5 shows the CEX-HPLC residuals and main effects plots for the second round of excipient screening.

[0018] Figure 6 shows the response surface optimization overlaid contour plots.

[0019] Detailed Description of the Invention

[0020] definition:

[0021] Hyaluronidases are a class of enzymes that are generally active under neutral or acidic conditions. Generally speaking, there are three major classes of hyaluronidases:

[0022] 1. Mammalian hyaluronidase (EC 3.2.1.35), which is an endo-β-N-acetylhexosaminidase with tetrasaccharides and hexasaccharides as major end products.

[0023] 2. Bacterial hyaluronidase (EC 4.2.99.1) degrades hyaluronan and, to varying degrees, chondroitin sulfate (CS) and dermatan sulfate (DS).

[0024] 3. Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, and crustaceans are endo-β-glucuronidases that hydrolyze β1-3 linkages to form tetrasaccharide and hexasaccharide end products.

[0025] The hyaluronidase present in the subcutaneous administration preparation according to the present application can be prepared by using recombinant DNA technology or extracted from animal tissue; the recombinant humanized hyaluronidase used in the formulation exemplified herein is rHuPH20, which can be prepared by using recombinant DNA technology or commercially available human hyaluronidase products.

[0026] Currently, there are many hyaluronidase products on the market. For example, animal-derived hyaluronidase products approved in the United States include Vitrase (sheep hyaluronidase), Hydase (bovine testicular hyaluronidase) and Amphadase (bovine hyaluronidase).

[0027] It is a recombinant human hyaluronidase for injection that was launched in 2005. Its amino acid sequence is known and is available under CAS registration number 757971-58-7.

[0028] A "biologically active fragment" of a given hyaluronidase is a fragment of the protein that retains its ability to hydrolyze hyaluronic acid. One skilled in the art can design truncations based on the domain structure of the hyaluronidase and test their ability to hydrolyze hyaluronic acid to obtain a suitable biologically active fragment.

[0029] In a broad sense, "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses intact antibodies / full-length antibodies, single antibody chains, or any antigen-binding fragment of an antibody (also referred to as an "antigen-binding portion"). When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" can be understood as the intact body relative to the "antigen-binding fragment / antigen-binding portion", and the two together correspond to the broad concept of antibody.

[0030] As used herein, the terms "antigen-binding fragment" or "antigen-binding portion" are used interchangeably and refer in particular to antibody fragments such as Fv, Fab, F(ab')2 or Fab', or any fragment that has been modified by chemical modification, such as the addition of poly(alkylene) glycols such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) or by incorporation into liposomes to increase half-life ("PEG" refers to polyethylene glycol), wherein the fragment has CD73 binding activity. Preferably, the functional fragment will consist of or comprise a portion of the heavy chain or light chain variable region of the antibody from which it is derived, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived. Such a functional fragment will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived. Examples of antigen-binding fragments include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments connected by a disulfide bridge at the hinge region (i.e., a dimer of Fab'); and (3) an Fv fragment having the VL and VH domains of a single arm of an antibody.

[0031] As used herein, the term "functional fragment" refers to a fragment that, in addition to retaining antigen-binding function (i.e., in addition to being an "antigen-binding fragment" or "antigen-binding portion"), also retains one or more other functions of an antibody (e.g., the function of mediating ADCC and / or CDC).

[0032] The term "humanized antibody" means an antibody obtained by grafting CDR sequences derived from another mammalian species, such as a mouse germline, onto human framework sequences. In order to retain binding affinity, some residues of the backbone (called FR) segment may be modified. Humanized antibodies or fragments thereof according to the present application may be prepared by techniques known to those skilled in the art.

[0033] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor individuals.

[0034] The term "Tonset" refers to the melting onset temperature.

[0035] The term "Tm" refers to melting temperature.

[0036] The term "SEC-HPLC" refers to size exclusion chromatography.

[0037] The term "CEX-HPLC" refers to cation exchange chromatography.

[0038] The term "Z-average" refers to the hydrodynamic radius.

[0039] The term "PDI" refers to polydispersity index.

[0040] The term "kD" refers to the interaction coefficient.

[0041] The term "A2" refers to the second virial coefficient.

[0042] The term "Tagg" refers to the aggregation temperature.

[0043] The term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show benefit to the individual to whom it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the individual condition and severity of the condition being treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and is relied upon by general practitioners and other physicians, generally taking into account the condition being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0044] The present application relates to anti-CD73 antibody pharmaceutical compositions.

[0045] In one aspect, the present application relates to a pharmaceutical composition comprising (i) an anti-CD73 antibody or an antigen-binding portion or functional fragment thereof, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.

[0046] The term "stabilizer" in the present application also encompasses viscosity reducing agents that function to reduce viscosity.

[0047] In some embodiments, the buffer is an organic acid buffer, including but not limited to acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, and glycylglycine.

[0048] In some embodiments, the histidine salt buffer is a buffer comprising histidine ions. Examples of histidine salt buffers include histidine-histidine hydrochloride (also referred to herein as "histidine hydrochloride"), histidine-acetate, histidine-phosphate, histidine-sulfate, and the like, wherein the histidine-acetate buffer is a mixture of histidine and acetic acid, and the histidine-histidine hydrochloride buffer is a mixture of histidine and histidine hydrochloride. Similarly, the succinate buffer can be a mixture of succinic acid and sodium succinate, and the citrate buffer can be a mixture of citric acid and sodium citrate.

[0049] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0050] In some embodiments, the stabilizer includes but is not limited to sugars (such as sucrose, trehalose), polyols (such as mannitol, sorbitol), and amino acids (L-serine, sodium glutamate, alanine, glycine, sarcosine, proline, methionine, etc.).

[0051] In some embodiments, the surfactant includes, but is not limited to, polysorbate 20 or polysorbate 80.

[0052] In some embodiments, the pH of the pharmaceutical composition is 4.0-7.4, preferably 5.0-6.9, more preferably 6-6.6; further preferably, the pH is about 5.5.

[0053] In some embodiments, the pharmaceutical composition of the present application further contains water.

[0054] In some embodiments, the pharmaceutical composition is isotonic (typically relative to plasma for clinical use) and thus also includes isotonic formulations, which are liquids or liquids reconstituted from solid forms, representing solutions with the same tonicity as some other solution (e.g., plasma) to which they are compared. Suitable isotonic agents include, but are not limited to, sodium chloride, potassium chloride, glycerol, glycine, proline, arginine hydrochloride, lysine, sucrose, trehalose, mannitol, sorbitol, and any combination thereof.

[0055] In some embodiments, the pharmaceutical composition of the present application comprises or has:

[0056] Anti-CD73 antibody at a concentration of 5-250 mg / ml, for example, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml, about 210 mg / ml, about 220 mg / ml, about 230 mg / ml, about 240 mg / ml or about 250 mg / ml;

[0057] Histidine-histidine hydrochloride at a concentration of 5-30 mM, e.g., about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM;

[0058] Polysorbate 20 or polysorbate 80 at a concentration of 0.002% (w / v) to 0.08% (w / v), for example, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.010%, 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07% or about 0.08%;

[0059] Sucrose at a concentration of 20-50 mg / ml, e.g., about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 50 mg / ml;

[0060] Sodium chloride at a concentration of 1-50 mg / ml, for example, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 50 mg / ml;

[0061] pH 5.0-7.0, e.g., about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0062] In some embodiments, the pharmaceutical compositions of the present application (eg, per unit dosage form) further comprise at least about 5 U to at least about 100,000 U of hyaluronidase.

[0063] In some embodiments, the pharmaceutical composition (e.g., per unit dosage form) comprises at least about 5 U, at least about 10 U, at least about 20 U, at least about 30 U, at least about 40 U, ​​at least about 50 U, at least about 75 U, at least about 100 U, at least about 200 U, at least about 300 U, at least about 400 U, at least about 500 U, at least about 750 U, at least about 1000 U, at least about 2000 U, at least about 3000 U, at least about 4000 U, At least about 5000 U, at least about 6000 U, at least about 7000 U, at least about 8000 U, at least about 9000 U, at least about 10,000 U, at least about 20,000 U, at least about 30,000 U, at least about 40,000 U, at least about 50,000 U, at least about 60,000 U, at least about 70,000 U, at least about 80,000 U, at least about 90,000 U, or at least about 100,000 U of hyaluronidase.

[0064] In some embodiments, the pharmaceutical composition (eg, per unit dosage form) comprises about 20,000 U of hyaluronidase.

[0065] In some embodiments, the hyaluronidase comprises a hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, any variants and any isoforms of the above.

[0066] In some embodiments, the hyaluronidase comprises rHuPH20 or a fragment (eg, a biologically active fragment) thereof.

[0067] In some embodiments, the hyaluronidase is a modified hyaluronidase variant comprising one or more amino acid substitutions relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or a fragment (e.g., a biologically active fragment) thereof.

[0068] In some embodiments, pharmaceutical composition comprises at least about 500U / mL to at least about 5000U / mL hyaluronidase.In some embodiments, pharmaceutical composition comprises at least about 1000U / ml, 1500U / mL, at least about 1600U / mL, at least about 1700U / mL, at least about 1800U / mL, at least about 1900U / mL, at least about 2000U / mL, at least about 2100U / mL, at least about 2200U / mL, at least about 2300U / mL, at least about 2400U / mL, at least about 2500U / mL, at least about 3000U / mL, at least about 3500U / mL, at least about 4000U / mL, at least about 4500U / mL or at least about 5000U / mL hyaluronidase.In some embodiments, pharmaceutical composition comprises the hyaluronidase of about 2000U / mL.

[0069] In some embodiments, the components of the pharmaceutical composition of the present application include: 40 mg / ml-250 mg / ml anti-CD73 antibody, 5-20 mM histidine-histidine hydrochloride, 1-10 mg / ml sodium chloride, 20-50 mg / ml sucrose, 0.002%-0.08% (w / v) polysorbate 80, pH 5.0-7.0.

[0070] In some embodiments, the pharmaceutical composition of the present application includes: 50 mg / ml anti-CD73 antibody, 10 mM histidine-histidine hydrochloride, 6 mg / ml sodium chloride, 25 mg / ml sucrose, 0.005% (w / v) polysorbate 80, pH 6.3±0.3.

[0071] In some embodiments, the pharmaceutical composition of the present application further contains water, such as water for injection.

[0072] In some embodiments, the pharmaceutical composition of the present application consists of 50 mg / ml anti-CD73 antibody, 10 mM histidine-histidine hydrochloride, 6 mg / ml sodium chloride, 25 mg / ml sucrose, 0.005% (w / v) polysorbate 80 and water for injection, pH 6.3.

[0073] In some embodiments, the pharmaceutical composition of the present application comprises: 100-250 mg / ml anti-CD73 antibody, 5-30 mM histidine-histidine hydrochloride, 100-200 mM proline, 5-20 mM methionine, 20-50 mg / ml sucrose, 0.01%-0.08% (w / v) polysorbate 80, 1000-3000 U / ml hyaluronidase, pH 5.0-7.0.

[0074] In some embodiments, the pharmaceutical composition of the present application comprises: 200 mg / ml anti-CD73 antibody, 20 mM histidine-histidine hydrochloride, 125 mM proline, 10 mM methionine, 40 mg / ml sucrose, 0.02% (w / v) polysorbate 80, 2000 U / ml hyaluronidase, pH 5.5.

[0075] In some embodiments, the pharmaceutical composition of the present application further contains water, such as water for injection.

[0076] In some embodiments, the pharmaceutical composition of the present application consists of 200 mg / ml anti-CD73 antibody, 20 mM histidine-histidine hydrochloride, 125 mM proline, 10 mM methionine, 40 mg / ml sucrose, 0.02% (w / v) polysorbate 80, 2000 U / ml hyaluronidase and water for injection, pH 5.5.

[0077] In some embodiments, the pharmaceutical composition is in the form of a liquid preparation, or in the form of a lyophilized preparation or a powder injection preparation prepared from a liquid preparation.

[0078] On the other hand, the present application also relates to a method for preparing the above-mentioned pharmaceutical composition, the method comprising:

[0079] 1) Calculate the amount of each excipient based on the required solution volume and the expected concentration of each excipient, accurately weigh them, prepare them with water for injection, and then sterile filter them through a 0.22 μm filter to obtain the excipient preparation solution;

[0080] 2) Using the ultrafiltration replacement method, the anti-CD73 antibody preparation liquid is replaced with the above-mentioned excipient preparation liquid, and the antibody concentration is adjusted to the desired concentration using the above-mentioned excipient preparation liquid depending on the antibody concentration after the exchange; the CD73 antibody preparation after the liquid exchange is sterile filtered through a 0.22 μm low protein binding filter, and then aseptically packaged into medium borosilicate glass tube injection bottles, and sealed with a combination of a film-coated rubber stopper and an aluminum cap.

[0081] In another aspect, the anti-CD73 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises three CDR regions (HCDR1, HCDR2, HCDR3) having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and / or the light chain comprises three CDR regions (LCDR1, LCDR2, LCDR3) having the amino acid sequences shown in SEQ ID NOs: 5, 6, and 7, respectively.

[0082] The heavy and light chain CDRs of this application are according to the Kabat definition.

[0083] In some embodiments, the heavy chain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:4, and / or the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8.

[0084] In some embodiments, the anti-CD73 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDR regions (HCDR1, HCDR2, HCDR3) having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and / or the light chain variable region comprises three CDR regions having the amino acid sequences shown in SEQ ID NOs: 5, 6, and 7, respectively.

[0085] In some embodiments, the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:9, and / or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:10.

[0086] In one embodiment, the present application also provides a lyophilized composition obtained by freeze-drying a composition in liquid form. The present application also provides a solution obtainable by dissolving (eg, with water) the lyophilized composition.

[0087] In some embodiments, the antibody is a monoclonal antibody or a bispecific antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a fully human antibody.

[0088] In some specific embodiments, the antibodies or functional fragments thereof of the present application have ADCC activity.

[0089] In some specific embodiments, the antibodies or functional fragments thereof of the present application have CDC activity.

[0090] In some specific embodiments, the antibody or functional fragment thereof of the present application is an IgG1κ antibody.

[0091] In some specific embodiments, the antibody or functional fragment thereof of the present application further comprises a human constant region.

[0092] In some specific embodiments, the antibody or functional fragment thereof of the present application further comprises a human IgG1 heavy chain constant region and a human κ light chain constant region.

[0093] In some specific embodiments, the antibody or functional fragment thereof of the present application further comprises an engineered Fc region, and the engineered Fc region enables the antibody to have enhanced ADCC and / or CDC effects.

[0094] In some embodiments, the anti-CD73 antibody or its antigen-binding portion is engineered to have weakened ADCC or CDC activity. In some embodiments, the anti-CD73 antibody comprises an engineered Fc region, and the engineered Fc region enables the antibody to have weakened ADCC and / or CDC effects. For the target CD73 and in vivo application of the antibody targeted by the present application, antibodies with weakened or even eliminated ADCC or CDC activity may be advantageous, because an overly strong Fc effect may affect or even weaken the efficacy of the antibody. Ways to weaken or eliminate the Fc effect are known in the art, for example, 1) amino acid residue mutation modification (mainly to weaken binding to related receptors), 2) glycosylation modification, 3) IgG4 antibody modification, etc. As a non-limiting example, the antibody has a human IgG1 heavy chain constant region and contains a mutation in one or more of L234, L235, P329, P331, wherein the residues are numbered according to the EU numbering system, e.g., the mutation is one or more of L234A, L235A, P329A, P331S.

[0095] On the other hand, the present application relates to an isolated nucleic acid molecule encoding the anti-CD73 antibody or its antigen-binding portion or functional fragment.

[0096] In another aspect, the present application relates to a vector (eg, an expression vector) containing the nucleic acid molecule.

[0097] An "expression vector" is a vector that includes one or more expression control sequences, which are DNA sequences that control and regulate the transcription and / or translation of another DNA sequence. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus.

[0098] In another aspect, the present application relates to a host cell containing the nucleic acid molecule or the vector.

[0099] In some embodiments, the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic host cell can be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, an insect cell such as S. frugiperda, a plant cell such as tobacco, or a mammalian cell such as BHK cells, CHO cells, COS cells, or myeloma cells.

[0100] On the other hand, the present application provides the anti-CD73 antibody or its antigen-binding portion or functional fragment, the nucleic acid molecule, the vector, the host cell for use in the preparation of a method for treating a tumor in an individual, particularly a tumor cell expressing CD73 (CD73 + The drug can be the pharmaceutical composition described above.

[0101] In another aspect, the present application provides a method for treating a tumor in an individual, particularly a tumor cell expressing CD73 (CD73 + ) tumor, the method comprising administering to the individual a therapeutically effective amount of the anti-CD73 antibody or its antigen-binding portion or the pharmaceutical composition described above.

[0102] In some embodiments, the tumor is a tumor cell that highly expresses CD73 (CD73 + In some embodiments, the tumors that highly express CD73 (CD73 + ) refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express CD73.

[0103] In some embodiments, the tumor is a solid tumor.

[0104] In some embodiments, the tumor is a hematological tumor.

[0105] In some embodiments, the tumor is a malignant tumor.

[0106] In some embodiments, the tumor is cancer.

[0107] In some embodiments, the tumor is selected from the group consisting of ovarian cancer, breast cancer, melanoma, colorectal cancer, gastric cancer, renal cancer, prostate cancer, pancreatic cancer, esophageal cancer, head and neck cancer, thyroid cancer, lung cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, squamous cell carcinoma, vulvar cancer, anal cancer, penile cancer, brain cancer, lymphoma (e.g., B-cell lymphoma), leukemia, and metastases of the above tumors.

[0108] In some embodiments, the pharmaceutical compositions of the present application can be delivered by any physiologically acceptable mode of administration, including but not limited to oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.

[0109] In some embodiments of the present application, the optimal formulation of the intravenous preparation was determined by screening and optimizing pH, buffer system, stabilizer, and surfactant. Such a preparation can fully prevent protein aggregation and degradation, thereby maintaining the biological activity of its effective components and is suitable for clinical use. In a specific embodiment, a high-concentration preparation containing 200 mg / ml anti-CD73 antibody (such as Huab1F2F9.15) was developed by screening and optimizing excipients such as pH, buffer system, stabilizer (including viscosity reducer), and surfactant. The preparation containing a high concentration of anti-CD73 antibody is stable, can fully prevent protein aggregation, and its stability is comparable to that of the intravenous preparation;

[0110] In specific embodiments, by examining the stability of a high-concentration anti-CD73 antibody combined with hyaluronidase complex formulation, it was demonstrated that the anti-CD73 antibody (e.g., Huab1F2F9.15) and hyaluronidase in the formulation were both stable, not prone to aggregation and degradation, safe, and highly effective, and could be used for subcutaneous injection, with good application prospects. Example:

[0111] Example 1: Acquisition of anti-CD73 humanized monoclonal antibodies

[0112] Healthy BALB / c mice were immunized with recombinant human CD73 protein (Arco, product number CD3-H52H7). After the initial immunization, booster immunizations were performed three times every 14 days. The peripheral serum of the mice was taken, and the mouse serum titer was detected by enzyme-linked immunosorbent assay (ELISA), and mice with high plasma antibody titers were selected for cell fusion. Three days before fusion, shock immunization was performed through the tail vein or abdominal cavity. On the day of fusion, the spleen of the mouse was taken and prepared into a single cell suspension. The mouse myeloma SP2 / 0 cells and spleen cells were mixed in a ratio of 1:2 and fused by electrofusion. The cells were cultured in a 37°C CO2 incubator. When the hybridoma cells grew to a certain amount, the hybridoma supernatant was screened by ELISA. Positive clones were selected and subcloned by limiting dilution. Finally, the monoclonal positive cells obtained were extracted from RNA, reverse transcribed and amplified by PCR to obtain mouse anti-CD73 antibodies.

[0113] The relevant mouse antibodies were humanized, specifically using Discovery Studio and Antibody Modeling performed homology modeling. Through structural simulation and rational design, we analyzed and obtained the humanized framework region that was closest to the mouse antibody framework region, ultimately obtaining the humanized antibody Huab1F2F9.15.

[0114] The antibody sequences are as follows: the underlined CDR sequences (determined and annotated according to the Kabat numbering system).

[0115] Heavy chain variable region (SEQ ID NO: 4):

[0116] SEQ ID NO.1:SYWMH (HCDR1)

[0117] SEQ ID NO.2:NINPSNGGTKYSQKFQG(HCDR2)

[0118] SEQ ID NO.3:RDYGSPSY(HCDR3)

[0119] Light chain variable region (SEQ ID NO.8)

[0120] SEQ ID NO.5:KASQDINSYLT(LCDR1)

[0121] SEQ ID NO.6: RTNILLD (LCDR2)

[0122] SEQ ID NO.7: LQYHEFPVT (LCDR3)

[0123] Heavy chain (SEQ ID NO: 9):

[0124] Light chain (SEQ ID NO: 10):

[0125] Example 2: Binding ability detection of humanized antibodies

[0126] The affinity of humanized antibody Huab1F2F9.15 to recombinant human CD73 protein (same as above) was determined by ELISA. The recombinant human CD73 protein was coated on the ELISA plate at a concentration of 1 μg / ml; 5-fold gradient dilutions of the antibody (0-66.7 nM) were added. The experimental results showed that its EC 50 Value (nM) = 0.011. The results show that the anti-CD73 antibody Huab1F2F9.15 has good antigen binding ability.

[0127] Example 3: In vitro inhibitory activity of humanized antibodies

[0128] CD73-expressing MDA-MB-231 cells were cultured at 2×10 5 / ml, 100μl / well was inoculated into a 96-well plate. The plate was incubated at 37°C for 1h, and then adenosine monophosphate (AMP) (1800μM) was added to each well, 50μl / well, and incubated at 37°C for 2 hours. The cells were centrifuged at 1000rpm for 5min, and 50μl of supernatant was transferred to a new 96-well plate, and 50μl of adenosine triphosphate (ATP) was added to make its final concentration 75μM. CellTiter-Glo reagent (Promega) was added at a 1:1 ratio to determine the cellular CD73 enzyme activity. The results showed that Huab1F2F9.15 can inhibit the CD73 enzyme activity on MDA-MB-231 cells, and the cellular enzyme activity inhibition IC 50 (nM) = 0.069. The results showed that the anti-CD73 antibody Huab1F2F9.15 had significant in vitro inhibitory activity against CD73.

[0129] Example 4: Evaluation of the biological activity of anti-CD73 antibodies in animals

[0130] The efficacy of Hab1F2F9.15, anti-PD-1 antibodies, and their combination were evaluated using BALB / c mice with human CD73 / PD1 / PDL1 knock-in and CT26 cells (colon cancer cell line) overexpressing human CD73 / PDL1. 6CT26-hCD73 / hPDL1 cells were subcutaneously inoculated into transgenic mice. 3 Around 6:00 p.m., mice were grouped according to tumor volume. The day of grouping was defined as D0, and dosing began on the day of grouping. The designated dose of antibody or PBS control was injected intraperitoneally twice a week. Mice were treated with anti-PD-1 antibody alone (1 mg / kg), anti-CD73 antibody alone (Hab1F2F9.15 and Oleclumab (MEDI9447); 15 mg / kg), and anti-PD-1 antibody plus anti-CD73 antibody (1 mg / kg + 15 mg / kg).

[0131] The results showed that the combination of Huab1F2F9.15 and anti-PD-1 antibody and MEDI9447 and anti-PD-1 antibody reduced tumor growth, resulting in 53.74% and 37.67% tumor inhibition, respectively. The combination of Hab1F2F9.15 and anti-PD-1 antibody was significantly better than the combination of MEDI9447 and anti-PD-1 antibody (Figure 1A). The results of individual tumor volumes are shown in Figure 1B (multiple lines represent different mice).

[0132] Example 5: Preparation and property testing of liquid formulations

[0133] 5.1 Preparation of intravenous formulations

[0134] Huab1F2F9.15 was prepared into various liquid formulations of pharmaceutical compositions described below, collectively referred to as SYCD73.

[0135] The amount of each excipient is calculated based on the required solution volume and the expected concentration of each excipient, accurately weighed, and prepared with water for injection. The excipient solution is then sterile-filtered through a 0.22 μm filter to obtain an excipient preparation liquid. The excipient preparation liquid is then used to replace the Huab1F2F9.15 antibody preparation liquid using an ultrafiltration replacement method. Depending on the antibody concentration after the exchange, the antibody concentration is adjusted to the desired concentration using the excipient preparation liquid. The Huab1F2F9.15 antibody preparation after the replacement liquid is sterile-filtered through a 0.22 μm low protein binding filter and aseptically dispensed into medium borosilicate glass injection vials. The vials are sealed with a film-coated rubber stopper and an aluminum-plastic combination cap to prepare the SYCD73 preparation.

[0136] 5.2 Preparation of Subcutaneous Formulations

[0137] To prepare the formulation solution, Huab1F2F9.15 is buffer exchanged with a pre-prepared subcutaneous formulation excipient (the general process is the same as 5.1), and concentrated by ultrafiltration to the desired antibody concentration or above when necessary. After ultrafiltration and concentration, the antibody concentration is adjusted to the desired concentration with the corresponding buffer, the volume of polysorbate 80 and rHuPH20 stock solution added is calculated based on the sample volume, and polysorbate 80 and / or rHuPH20 are added under sterile conditions. All preparations are sterile filtered through a 0.22μm low protein binding filter. Under sterile conditions, the mixture is dispensed into borosilicate glass injection bottles and sealed with a film-coated rubber stopper and an aluminum-plastic combination cap.

[0138] 5.3 General test methods for preparation properties

[0139] The detection methods involved in the following examples are described in a unified manner below. If there are other detection conditions, parameters or step adjustments in the examples, they will be specifically noted in the examples.

[0140] (1) UV spectrophotometer method;

[0141] Protein content was determined using a Nanodrop micro-spectrophotometer. Blank solution was used for sample preparation for blank subtraction, and the UV absorption at 280 nm was corrected by light scattering at 350 nm. Protein concentration was calculated according to Formula 1, where the extinction coefficient ε is 1.468 (L / g.cm -1 ).

[0142] (2) Differential Scanning Calorimetry (DSC):

[0143] The thermal stability of the formulation was analyzed using a differential scanning calorimeter (DSC). The test sample was diluted with the same preparation solution to an antibody concentration of 1 mg / ml. 350 μl of each test sample and corresponding preparation solution were added to the corresponding positions of a 96-well plate. The 96-well plate was placed in the sample well, and the sample well temperature was set to 15°C. Instrument parameters were set as follows: start temperature 20°C, end temperature 100°C, and heating rate 90°C / min.

[0144] (3) Size Exclusion Chromatography (SEC-HPLC):

[0145] Soluble high molecular weight species (HMWS) and low molecular weight species (LMWS) in the formulation were detected using size exclusion chromatography (SEC-HPLC). This method was performed on an Agilent 1260 instrument equipped with a TOSOH TSKgel G3000Wxl column. 200 mM potassium phosphate and 245 mM potassium chloride at pH 6.2 were used as the mobile phase. Intact monomers, aggregates, and fragments were separated by isocratic elution, and detection was performed at a wavelength of 280 nm.

[0146] (4) Cation exchange chromatography (CEX-HPLC):

[0147] Cation exchange chromatography (CEX-HPLC) was used to detect the acidic peak, main peak, and alkaline peak ratios in the preparation. This method was implemented on an Agilent 1260 instrument equipped with a YMC BioPro IEX SF column. A 20 mM 2-morpholinolineethanesulfonic acid (MES) solution with a pH of 6.10 was used as mobile phase A, and a 20 mM 2-morpholinolineethanesulfonic acid and 500 mM NaCl solution was used as mobile phase B. The main peak, acidic peak, and alkaline peak were separated by gradient elution and detected at a wavelength of 280 nm.

[0148] 5) Non-reducing capillary electrophoresis-sodium dodecyl sulfate (NR-CE-SDS)

[0149] Soluble high molecular weight species (HMWS) and low molecular weight species (LMWS) in the formulations were determined using non-reducing capillary electrophoresis-sodium dodecyl sulfate (NR-CE-SDS). After heat denaturation of the sample in the presence of sodium dodecyl sulfate (SDS), the method was performed on a capillary electrophoresis instrument (MauriceFlex) equipped with non-coated capillaries. Injection: 4600 V, 20 s; separation: 5750 V, 40 min; detection was performed using a PDA detector at a wavelength of 220 nm.

[0150] (6) Isothermal stability test

[0151] Freshly prepared samples were added to a 96-well plate and treated at 57°C for 16.5 hours (based on DSC results). Changes in sample stability during and after the treatment were evaluated. During the DLS test, Z-average monitoring was performed every half hour. Changes in the main peak purity of the samples were also tested before and after the test using SEC-HPLC, CEX-HPLC, and NR-CE-SDS.

[0152] (7) Flow Imaging Particle Analysis System (FlowCam) - Insoluble Particle Detection:

[0153] Subvisible particles in the sample were analyzed using a flow imaging particle analysis system (FlowCam). After autofocusing using a 15 μm standard bead solution, the sample to be tested was gently mixed while avoiding bubbles, and 150 μl was pipetted into the injection port for analysis. Instrument parameters were set as follows: sample volume 150 μl, flow rate 150 μl / min.

[0154] (8) Dynamic Light Scattering (DLS):

[0155] The colloidal stability of the samples was analyzed using a dynamic light scattering (DLS) instrument. After preheating the instrument for 30 minutes, 30 μl of sample was added to a 384-well sample plate. The plate was sealed with a transparent sealing film. The plate was centrifuged at 2000-3000 rpm until no bubbles were observed. The plate was then placed in the sample well for testing.

[0156] (9) Microfluidic Viscometer (mircoVISC):

[0157] Viscosity of the formulation was measured using a microfluidic viscometer (mircoVISC). After preheating the viscometer to the test temperature, the chip was cleaned 2-3 times. Before testing, the sample was filtered through a 0.22 μm filter and centrifuged for 1-2 minutes to remove air bubbles. An appropriate volume was collected and placed in the instrument for testing.

[0158] (10) Turbidimetric method for detecting hyaluronidase rHuPH20 activity;

[0159] Hyaluronidase activity was assayed using a turbidimetric assay. A low-protein-adsorbed 96-well sample plate containing the test sample and standard, and a 96-well plate containing hyaluronic acid working solution were incubated at 37°C. The test sample and standard were transferred from the sample plate to the substrate plate for reaction. The serum working solution was then added to the substrate plate to terminate the reaction. The absorbance was measured at 640 nm, and the activity of the test sample was calculated based on the standard curve.

[0160] Example 6: pH and buffer system screening

[0161] pH value is an important factor affecting the stability of biological product preparations. It regulates the surface charge distribution of proteins, affects the forces between and within protein molecules, and also affects the conformational stability, colloidal stability, and chemical stability of proteins. The buffer system also has an important influence on maintaining the pH stability of the preparation and the stability of the protein.

[0162] This embodiment first studies the effect of pH value and buffer system on the preparation. The preparation is formulated with antibody (Huab1F2F9.15) and buffer, without other excipients. Specifically, the pH investigation range is 5.0-7.5, and the buffer system includes acetate buffer system, histidine-histidine hydrochloride buffer system, citrate buffer system and phosphate buffer system, and the concentration is 20mM. A liquid preparation with an antibody concentration of 50mg / ml was prepared, and stability was studied under conditions such as high temperature (50°C), vibration (200rpm, 25°C) and freeze-thaw (-80°C to room temperature). Thermal stability, molecular size variant stability (SEC-HPLC) and charge variant stability (CEX-HPLC) were investigated, and the results are shown in Tables 1 to 5 below.

[0163] Taking into account the results in Tables 1 to 5, it was concluded that the 20 mM acetate buffer system at pH 5.0-6.0 and the histidine-histidine hydrochloride buffer system achieved relatively excellent stability.

[0164] Table 1 DSC test results

[0165] Note: Immediately dilute to an antibody concentration of 1 mg / ml for detection after preparation

[0166] Table 2 High temperature stability SEC-HPLC main peak results

[0167] Note: A- represents acetate buffer system; H- represents histidine-histidine hydrochloride buffer system; C- represents citrate buffer system; P- represents phosphate buffer system; the numbers represent pH values.

[0168] Table 3 CEX-HPLC results at high temperature for one week

[0169] Table 4 SEC-HPLC results of stability after one week of oscillation

[0170] Table 5 SEC-HPLC results of stability after 3 freeze-thaw cycles

[0171] Example 7: Excipient Screening

[0172] Based on the analysis of the results of Example 6, the main instability of the anti-CD73 antibody pharmaceutical composition is manifested in a significant increase in the acid peak of CEX-HPLC under high temperature conditions, as well as the generation of polymers and fragments. The increase in the CEX-HPLC acid peak may be caused by chemical instability such as oxidation and / or deamidation reactions, which can also cause the generation of polymers and fragments. Therefore, Minitab software was used to perform a DOE screening test on excipients such as sugars (sucrose, trehalose), alcohols (sorbitol), amino acids (methionine), salts (sodium chloride), and acetate buffer systems and histidine buffer systems based on the formula of Example 6 (antibody concentration of 50 mg / ml).

[0173] The first round of excipient screening mainly included: sucrose (0, 80 mg / ml, shown as Suc in Figure 2), sorbitol (0, 40 mg / ml, shown as Sor in Figure 3), methionine (0, 4 mg / ml, the results are not shown in the attached figure), and sodium chloride (0, 9 mg / ml). It was carried out in an acetate buffer system with a pH of 5.0-6.0, with Tonset and CEX-HPLC main peak purity as response factors, respectively. When Tonset is used as the response factor, it can be seen from Figure 2 that pH, sodium chloride and sucrose have a significant effect on the thermal stability of Huab1F2F9.15, among which pH and sucrose are positively correlated, and sodium chloride is negatively correlated. When the purity of the main peak of CEX-HPLC is used as the response factor, it can be seen from Figure 3 that pH, sodium chloride and sorbitol are significant influencing factors, among which pH and sodium chloride are positively correlated, and sorbitol is negatively correlated. Overall: ① Within the pH range of 5.0-6.0, both Tonset and CEX-HPLC main peak purity were significantly affected, and both were positively correlated. ② Sodium chloride was a significant factor affecting both Tonset and CEX-HPLC main peak purity, with a negative correlation for Tonset and a positive correlation for CEX-HPLC main peak purity. Analysis of pH and buffer system screening tests revealed that the primary instability of Huab1F2F9.15 was the decrease in main peak purity in CEX-HPLC detection. Therefore, the sodium chloride range was selected based on its effect on CEX-HPLC main peak purity. ③ Sucrose and sorbitol: Sucrose was a significant factor affecting Tonset and had a positive correlation, but had no significant effect on CEX-HPLC main peak purity. Sorbitol was a significant factor affecting both Tonset and CEX-HPLC main peak purity, with a positive correlation for Tonset and a negative correlation for CEX-HPLC main peak purity. Therefore, sucrose was selected as the stabilizer for Huab1F2F9.15. ④Methionine in the range of 0-4 mg / ml has no significant impact on the purity of the Tonset and CEX-HPLC main peaks. Therefore, it was not added during the screening and optimization of intravenous formulations (it was subsequently added and tested in the formulation of high-concentration subcutaneous formulations for other purposes).

[0174] Based on the first round of excipient screening, it was found that pH had a significant positive correlation with the purity of the CEX-HPLC main peak. Considering the low optimal buffering range of the acetate buffer system, a fractional factorial experimental design was used to screen the formulation composition of the histidine-histidine hydrochloride buffer system (0, 20mM), acetate buffer system (0, 20mM), trehalose (0, 50mg / ml), sodium chloride (0, 9mg / ml), and pH (5.0, 6.0). Tonset and CEX-HPLC main peak purity were used as response factors, respectively. When Tonset was used as the response factor, as can be seen from Figure 4, pH, histidine-histidine hydrochloride buffer system, and sodium chloride were significantly influencing factors, with pH showing a positive correlation and histidine-histidine hydrochloride buffer system and sodium chloride showing a negative correlation. When the purity of the CEX-HPLC main peak was used as the response factor, Figure 5 shows that pH, histidine-histidine hydrochloride buffer system, and acetate buffer system were significant influencing factors, with pH showing a positive correlation and histidine-histidine hydrochloride buffer system and acetate buffer system showing negative correlations. Overall, ① within the pH range of 5.0-6.0, the histidine-histidine hydrochloride buffer system was a significant factor influencing both Tonset and the purity of the CEX-HPLC main peak, with both positive correlations. ② The histidine-histidine hydrochloride buffer system was a significant factor influencing both Tonset and the purity of the CEX-HPLC main peak, with both negative correlations. The acetate buffer system was negatively correlated with the purity of the CEX-HPLC main peak and had no significant effect on Tonset. Considering the significant positive correlation between pH and the purity of the Tonset and CEX-HPLC main peaks, and the optimal pH ranges for the histidine-histidine hydrochloride and acetate buffer systems, the histidine-histidine hydrochloride buffer system was selected as the buffer system for Huab1F2F9.15 in this experiment, with a concentration of 10 mM.

[0175] Example 8 Determination of surfactant addition amount

[0176] Protein drugs are prone to aggregation and adsorption during storage. The addition of surfactants can reduce aggregation during stirring, shaking, and freeze-thaw processes, preventing protein adsorption to container surfaces. Based on a previously optimized pH and buffer system (histidine-histidine hydrochloride buffer, 10 mM), samples were prepared with the addition of various concentrations of polysorbate 80 (PS80) (0.005%, 0.01%, and 0.02%). Stability studies were conducted under vortexing (Shanghai Hanno XH-D, intensity 5, 2 hours) and freeze-thaw cycles (-80°C to room temperature, 5 cycles). Subvisible particles were detected using a microfluidics imaging particle analyzer (FlowCam), and molecular size variants were detected using SEC-HPLC.

[0177] Under vortex conditions, the insoluble particle detection results (Table 6) show that compared with time 0, the total number of 10 μm+ insoluble particles after vortexing increased by approximately 1,700 times when no polysorbate 80 was added. After adding different concentrations of polysorbate 80, the total number of 10 μm+ insoluble particles after vortexing decreased by more than 99.8% compared with no polysorbate 80 addition. Overall, the protective effects of 0.005%, 0.01%, and 0.02% polysorbate 80 were basically the same. The SEC-HPLC results (Table 7) show that there was no significant difference in the purity of the SEC-HPLC main peak after vortexing when different concentrations of polysorbate 80 were added.

[0178] Under freeze-thaw conditions, it can be seen from the insoluble particle detection results (Table 8) that when the addition amount of polysorbate 80 is 0.005%, after five repeated freeze-thaw cycles, the total number of 10μm+ insoluble particles is basically the same as the 0 point. When no polysorbate 80 is added and the addition amount is 0.01% and 0.02%, the total number of 10μm+ insoluble particles increases significantly. Therefore, with respect to insoluble particles, the Huab1F2F9.15 formulation provides good protection after five repeated freeze-thaw cycles at 0.005% polysorbate 80. From the SEC-HPLC results (Table 9), it can be seen that there is no significant difference in the purity of the SEC-HPLC main peak after repeated freeze-thaw cycles when different concentrations of polysorbate 80 are added.

[0179] Therefore, considering the results of vortex and freeze-thaw tests, and given that 0.005% polysorbate 80 can effectively reduce the formation of subvisible particles without affecting the purity of the SEC-HPLC main peak, it was determined that 0.005% polysorbate 80 would be used as a surfactant in subsequent tests.

[0180] Table 6 Vortex test results of insoluble particles

[0181] Table 7 Vortex test SEC-HPLC test results

[0182] Table 8 Results of insoluble particles detected in freeze-thaw test

[0183] Table 9 SEC-HPLC test results of freeze-thaw test

[0184] Example 9 Response surface optimization experiment

[0185] Based on pH, buffer system and excipient screening, Minitab software was used to perform Box-Behnken experimental design to further optimize the formula composition.

[0186] Using a 10mM histidine-histidine-HCl buffer system as the base solution, the pH (5.5-6.5), sucrose (20-40 mg / ml), and sodium chloride (3-6 mg / ml) were optimized by examining thermal stability, size variant stability (SEC-HPLC), and charge variant stability (CEX-HPLC) at high temperature (50°C). Higher Tm, CEX-HPLC main peak purity, and SEC-HPLC main peak purity indicate more stable Huab1F2F9.15 formulations. Therefore, these response factors were optimized. Osmolarity, which was required to be isotonic with human plasma, was optimized. The resulting overlay contour plot is shown in Figure 6. Response Optimizer was used to optimize each response, resulting in the optimal formulation composition: pH 6.3, sodium chloride concentration 6 mg / ml, and sucrose concentration 25 mg / ml.

[0187] In summary, the optimized formula of the anti-CD73 antibody intravenous preparation pharmaceutical composition is 50 mg / ml anti-CD73 antibody, 10 mM histidine-histidine hydrochloride, 6 mg / ml sodium chloride, 25 mg / ml sucrose, 0.005% (w / v) polysorbate 80, pH 6.3.

[0188] Example 10 pH and buffer system screening of high concentration formulation (150 mg / ml anti-CD73 antibody)

[0189] Based on the experience in developing SYCD73 intravenous formulations, we screened 20mM histidine-histidine hydrochloride and acetate buffer systems for high-concentration formulations (suitable for subcutaneous injection) with a pH range of 5.0-6.0. Huab1F2F9.15 was replaced with the corresponding buffer systems, and the antibody concentration was adjusted to 150mg / ml. DSC, DLS, viscosity, SEC-HPLC, and CEX-HPLC were performed immediately after formulation. The results are shown in Tables 10 to 12.

[0190] Taking into account the results in Tables 10 to 12, 20 mM histidine-histidine hydrochloride at pH 5.5 was determined as the basic buffer system for the high-concentration formulation of Huab1F2F9.15.

[0191] Table 10 DSC results of pH and buffer system screening test

[0192] Table 11 DLS results of pH and buffer system screening test

[0193] Table 12 pH, buffer system screening test viscosity, SEC-HPLC and CEX-HPLC results

[0194] Example 11 Screening of viscosity-reducing excipients for high-concentration formulations (antibody concentration 200 mg / ml)

[0195] Based on the pH and buffer system screening, a 20mM, pH 5.5 histidine-histidine hydrochloride buffer system was selected as the basic system, and five viscosity-reducing excipients, proline, lysine hydrochloride, arginine hydrochloride, glycine, and sodium chloride, were screened. Each excipient had three concentration ranges of 50mM, 100mM, and 150mM. Huab1F2F9.15 was ultrafiltered into the above solution system, and the antibody concentration was adjusted to approximately 200mg / ml. Viscosity, DSC, and DLS tests were performed immediately after preparation. DLS, SEC-HPLC, CEX-HPLC, and NR-CE-SDS tests were performed in isothermal stability tests (all group results are not shown). The results are shown in Tables 13 to 17.

[0196] Taking into account the results in Tables 13 to 17, it was determined that glycine and proline were ideal viscosity-reducing excipients in the high-concentration formulation of Huab1F2F9.15.

[0197] Table 13 DSC results of viscosity-reducing excipient screening test

[0198] Table 14 DLS results of viscosity-reducing excipient screening test

[0199] Table 15 Viscosity test results of viscosity-reducing excipient screening test

[0200] Table 16 Isothermal stability test DLS detection

[0201] Table 17 Isothermal stability test results of related impurities

[0202] Note: "After the test" is 57℃, and the test is carried out after 16.5 hours

[0203] Example 12 Optimization of viscosity-reducing excipient concentration for high-concentration formulation (antibody concentration 200 mg / ml)

[0204] Based on the results of Example 11, 100 mM and 150 mM proline and 150 mM glycine can effectively reduce the viscosity of the formulation. In this example, the concentration range of proline and glycine was optimized, Huab1F2F9.15 was replaced into the above solution system, and the antibody concentration was adjusted to approximately 200 mg / ml. Viscosity, DSC, and DLS tests were performed immediately after formulation. SEC-HPLC and CEX-HPLC tests were performed in the isothermal stability test (57°C, 16.5 hours) (detection conditions were the same as in Example 11). The results are shown in Tables 18 to 21.

[0205] Based on the results in Tables 18 to 21, it was determined that in the high-concentration formulation of Huab1F2F9.15, the optimal viscosity-reducing concentration of proline was 125 mM, and the optimal viscosity-reducing concentration of glycine was 150 mM.

[0206] Table 18 DSC results of viscosity-reducing excipient concentration optimization test

[0207] Table 19 DLS results of viscosity-reducing excipient concentration optimization test

[0208] Table 20 Viscosity test results of viscosity-reducing excipient concentration optimization test

[0209] Table 21 Isothermal stability (57°C, 16.5h) test results of related impurities

[0210] Example 13 High Concentration Formulation (Antibody Concentration 200 mg / ml) Stabilizer Screening

[0211] Based on the pH, buffer system, and viscosity-reducing excipient screening performed in Examples 10-12, 20 mM histidine-histidine hydrochloride, 150 mM glycine, pH 5.5 was used as the base solution to investigate the effects of 10 mM methionine (Met) and 70 mg / ml sucrose (Sucrose) on the stability and viscosity of Huab1F2F9.15. The effects of 40 mg / ml sucrose (Sucrose) and 40 mg / ml trehalose (Trehalose) on the stability and viscosity of Huab1F2F9.15 were then investigated (the test conditions were the same as in Example 11). The results are shown in Tables 22 to 26.

[0212] Based on the results of Tables 22 to 26, 10 mM methionine and 40 mg / ml sucrose were determined as stabilizers for the high-concentration formulation of Huab1F2F9.15.

[0213] Table 22 DSC results of stabilizer screening test

[0214] Table 23 DLS results of stabilizer screening test

[0215] Table 24 Viscosity test results of stabilizer screening test

[0216] Table 25 Stabilizer Screening Test Product Related Impurity Detection Results (Isothermal Stability Test (57°C, 16.5h))

[0217] Table 26 Test results of the effects of 40 mg / ml sucrose and 40 mg / ml trehalose on viscosity and stability

[0218] Example 14 Determination of surfactant concentration in high concentration formulation (antibody concentration 200 mg / ml)

[0219] Taking into account the effect of sucrose concentration on viscosity, in order to balance the viscosity and stability of the solution, 20 mM histidine-histidine hydrochloride, 125 mM proline, pH 5.5, 10 mM methionine and 40 mg / ml sucrose were used as the base solution. The stability of the Huab1F2F9.15 formulation with different contents of polysorbate 80 was investigated under vortexing (Shanghai Hanno XH-D, intensity 3, 2 hours) and freeze-thaw (-80°C to room temperature, 10 times) conditions. The results are shown in Tables 27 to 28.

[0220] Based on the results in Tables 27 and 28, it was determined that the optimal addition amount of the surfactant polysorbate 80 in the high-concentration formulation of Huab1F2F9.15 was 0.02%.

[0221] Table 27 Vortex test (Shanghai Hanno XH-D, intensity 3, 2 hours) SEC-HPLC test results

[0222] Table 28 SEC-HPLC test results of freeze-thaw test (-80°C to room temperature, 10 times)

[0223] Example 15 Accelerated Stability Study of Two Formulations of High Concentration Formulations (Antibody Concentration 200 mg / ml)

[0224] Accelerated (storage at 40°C) stability studies were performed on two formulations derived from the previous screening work.

[0225] Formulation 1: 20 mM histidine-histidine hydrochloride, 150 mM glycine, 10 mM methionine, 40 mg / ml sucrose, pH 5.5;

[0226] Formulation 2: 20 mM histidine-histidine hydrochloride, 125 mM proline, 10 mM methionine, 40 mg / ml sucrose, pH 5.5.

[0227] Thermal stability, colloidal stability, size variant stability (SEC-HPLC), and charge variant stability (CEX-HPLC) were investigated, and the results are shown in Tables 29 to 30. The results show that both formulations performed excellently in the accelerated stability tests.

[0228] Table 29 DSC, DLS and viscosity test results (immediately tested after preparation)

[0229] Table 30 40℃ accelerated stability test results

[0230] Example 16 Stability Study of High Concentration Antibody Combined with rHuPH20 Formulation at Room and High Temperature (40°C)

[0231] Formulation A: 200 mg / ml anti-CD73 antibody (Huab1F2F9.15), 20 mM histidine-histidine hydrochloride, 150 mM glycine, 10 mM methionine, 40 mg / ml sucrose, 2000 U / ml rHuPH20, pH 5.5.

[0232] Table 31 Stability data of formula A

[0233] Formulation B: 200 mg / ml anti-CD73 antibody (Huab1F2F9.15), 20 mM histidine-histidine hydrochloride, 125 mM proline, 10 mM methionine, 40 mg / ml sucrose, 2000 U / ml rHuPH20, pH 5.5.

[0234] Table 32 Stability data of formulation B

[0235] The stability studies of the two formulations above indicate that the addition of 2000 U / ml rHuPH20 to the formulation has no effect on the purity of the Huab1F2F9.15 antibody, nor does the antibody have an effect on hyaluronidase activity. Furthermore, both rHuPH20 and the antibody are relatively stable in the two formulations above.

[0236] Example 17 Study on the Oscillation and Freeze-Thaw Stability of High-Concentration Antibody Combined with rHuPH20 Formulation

[0237] Formula C: 200 mg / ml anti-CD73 antibody (Huab1F2F9.15), 20 mM histidine-histidine hydrochloride, 125 mM proline, 10 mM methionine, 40 mg / ml sucrose, 2000 U / ml rHuPH20, 0.02% polysorbate 80, pH 5.5. Formula C is based on Formula B, but polysorbate 80 stock solution is added under sterile conditions to a final concentration of 0.02%. The stability of Formula C samples was investigated under shaking (200 rpm, 5°C) and freeze-thaw (-80°C to room temperature) conditions.

[0238] Table 33 Stability results of oscillation for 1 week

[0239] Table 34 Stability results after 10 freeze-thaw cycles

[0240] The results of oscillation stability and freeze-thaw stability tests showed that the addition of 0.02% polysorbate 80, followed by oscillation for one week and repeated freezing and thawing for 10 times, had no effect on the activity of rHuPH20 and the purity of Huab1F2F9.15.

[0241] The above description is merely a specific embodiment, which is intended to be illustrative and not limiting of the combination of features necessary to implement the present application. The titles provided are not intended to limit the various embodiments of the present application. Terms such as "comprise," "contain," and "include" are not intended to be limiting. In addition, unless otherwise indicated, plural forms are included when no numeral modifier is present, and "or" and "or" mean "and / or." Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0242] All disclosures and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the application, the multiple modifications and variants of the described method and composition of the application are apparent to those skilled in the art. Although the application has been described by specific preferred embodiments, it should be understood that the application for which protection is sought should not be unduly limited to these specific embodiments. In fact, those multiple variants of the described pattern that are apparent to those skilled in the relevant art are intended to be included in the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising (i) an anti-CD73 antibody or an antigen-binding portion or functional fragment thereof, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.

2. The pharmaceutical composition of claim 1, wherein the anti-CD73 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDR regions having amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, and / or the light chain variable region comprises three CDR regions having amino acid sequences as shown in SEQ ID NOs: 5, 6 and 7, respectively.

3. The pharmaceutical composition of claim 2, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO:4, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and / or The heavy chain of the anti-CD73 antibody comprises the amino acid sequence shown in SEQ ID NO:9 or consists of the amino acid sequence shown in SEQ ID NO:9, and / or the light chain of the anti-CD73 antibody comprises the amino acid sequence shown in SEQ ID NO:10 or consists of the amino acid sequence shown in SEQ ID NO:

10.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the buffer is an organic acid buffer, for example, the buffer is selected from one or more of acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, for example, acetate or histidine (e.g., histidine-histidine hydrochloride).

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the stabilizer is selected from one or more of sucrose, trehalose, mannitol, sorbitol, L-serine, sodium glutamate, alanine, glycine, sarcosine, proline, and methionine, for example, sucrose, methionine, glycine, proline, or any combination thereof.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the surfactant is selected from polysorbate 20 or polysorbate 80.

7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising an isotonic agent, wherein the isotonic agent is selected from one or more of sodium chloride, potassium chloride, glycerol, amino acids and sugars.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pharmaceutical composition comprises: 40mg / ml-250mg / ml of the anti-CD73 antibody, 5-20mM Histidine-Histidine HCl, 1-10mg / ml sodium chloride, 20-50 mg / ml sucrose, and 0.002%-0.08% (w / v) polysorbate 80; and the pharmaceutical composition has a pH of 5.0-7.

0.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises: 50 mg / ml of the anti-CD73 antibody, 10mM Histidine-Histidine HCl, 6mg / ml sodium chloride, 25 mg / ml sucrose, and 0.005% (w / v) polysorbate 80; and the pharmaceutical composition has a pH of 6.3±0.

3.

10. The pharmaceutical composition of any one of claims 1-8, further comprising hyaluronidase.

11. The pharmaceutical composition of claim 10, wherein the hyaluronidase is selected from the group consisting of rHuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof (eg, biologically active fragments).

12. The pharmaceutical composition of claim 11, wherein the hyaluronidase is rHuPH20.

13. The pharmaceutical composition of any one of claims 10-12, wherein the pharmaceutical composition comprises: 100-250 mg / ml of the anti-CD73 antibody, 5-30mM Histidine-Histidine HCl, 100-200mM proline, 5-20 mM methionine, 20-50mg / ml sucrose, 0.01%-0.08% (w / v) polysorbate 80, and 1000-3000 U / ml hyaluronidase; and the pharmaceutical composition has a pH of 5.0-7.

0.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition comprises: 200 mg / ml of the anti-CD73 antibody, 20mM Histidine-Histidine HCl, 125 mM proline, 10 mM methionine, 40mg / ml sucrose, 0.02% (w / v) polysorbate 80, and 2000U / ml hyaluronidase; and the pharmaceutical composition has a pH of 5.

5.

15. The pharmaceutical composition of any one of claims 1-14, for use in treating a tumor in an individual, such as a tumor that highly expresses CD73.

16. The pharmaceutical composition of claim 15, wherein the tumor that highly expresses CD73 is selected from: ovarian cancer, breast cancer, melanoma, colorectal cancer, gastric cancer, renal cancer, prostate cancer, pancreatic cancer, esophageal cancer, head and neck cancer, thyroid cancer, lung cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, squamous cell carcinoma, vulvar cancer, anal cancer, penile cancer, brain cancer, lymphoma, leukemia, and metastases of the above tumors.

17. Use of the pharmaceutical composition of any one of claims 1-14 in the preparation of a medicament for treating a tumor in an individual, such as a tumor that highly expresses CD73.

18. The use of claim 17, wherein the tumor with high expression of CD73 is selected from: ovarian cancer, breast cancer, melanoma, colorectal cancer, gastric cancer, renal cancer, prostate cancer, pancreatic cancer, esophageal cancer, head and neck cancer, thyroid cancer, lung cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, squamous cell carcinoma, vulvar cancer, anal cancer, penile cancer, brain cancer, lymphoma, leukemia, and metastases of the above tumors.

19. A method for treating a tumor in an individual, such as a tumor that highly expresses CD73, the method comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of any one of claims 1-14.

20. The method of claim 19, wherein the tumor that highly expresses CD73 is selected from: ovarian cancer, breast cancer, melanoma, colorectal cancer, gastric cancer, renal cancer, prostate cancer, pancreatic cancer, esophageal cancer, head and neck cancer, thyroid cancer, lung cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, squamous cell carcinoma, vulvar cancer, anal cancer, penile cancer, brain cancer, lymphoma, leukemia, and metastases of the above tumors.

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