Anti-CD112R antibody pharmaceutical composition and application thereof

Abstract

Provided are a stable anti-CD112R antibody pharmaceutical composition and a use thereof. The pharmaceutical composition contains a buffer solution and an anti-CD112R antibody or an antigen binding fragment thereof, wherein the concentration of the anti-CD112R antibody or the antigen binding fragment thereof is about 1 to 100 mg / mL. All the components of the developed antibody preparation interact and cooperate with one another, a storage environment suitable for long-term storage is provided for the anti-CD112R monoclonal antibody, and the antibody preparation has high stability and strong tolerance to high temperature, shaking and repeated freezing and thawing, and can block the interaction of CD112 / CD112R, improve the activation ability of T cells, mobilize the immune system and improve the anti-CD112R activity. And a stronger tumor killing effect is achieved.

Description

Anti-CD112R antibody pharmaceutical composition and use thereof Technical Field

[0001] The present invention relates to the field of therapeutic pharmaceutical compositions, and in particular to anti-CD112R antibody pharmaceutical compositions and uses thereof. Background Art

[0002] The immune system is a highly complex system composed of many cell types, including but not limited to T cells, B cells, natural killer cells, antigen-presenting cells, dendritic cells, monocytes, and macrophages. These cells possess a complex and delicate system to control their interactions and responses. Cells utilize activation and inhibition mechanisms and feedback loops to maintain control of responses and not allow the negative consequences of uncontrolled immune responses (e.g., autoimmune diseases) to occur.

[0003] CD112R (PVRIG), also known as poliovirus receptor-associated immunoglobulin domain-containing protein, Q6DKI7, or C7orf15, is a 326-amino acid transmembrane domain protein with a signal peptide (spanning amino acids 1 to 40), an extracellular domain (spanning amino acids 41 to 171), a transmembrane domain (spanning amino acids 172 to 190), and a cytoplasmic domain (spanning amino acids 191 to 326). CD112R binds to poliovirus receptor-related protein 2 (PVLR2, also known as nectin-2, CD112, or herpesvirus entry mediator B (HVEB), a human plasma membrane glycoprotein), the binding partner of CD112R.

[0004] Administration of anti-CD112R immunotherapy offers the opportunity to increase, enhance, and sustain immune responses. CD112R is an inhibitory receptor expressed primarily by T cells and NK cells, competing with the activating receptor CD226 for binding to CD112. CD112 interacts with CD112R with higher affinity than with CD226, effectively modulating CD226-mediated cell activation. Anti-CD112R antibodies that block the interaction with CD112 directly limit inhibitory signaling downstream of CD112R while promoting greater immune cell activation by increasing the interaction of CD226 with CD112.

[0005] Treatment with anti-CD112R antibodies provides an opportunity to downregulate inhibitory signals that may occur when CD112R-expressing immune cells bind to CD112 on tumor cells and / or myeloid cells within the tumor microenvironment and may enhance, increase, and sustain anti-tumor immune responses.

[0006] Currently, the only anti-CD112R monoclonal antibody is SRF813 (Surface Oncology), a preclinical trial, and the need for more novel anti-CD112R antibodies remains. However, antibody drugs are large in molecular weight and complex in structure, making them susceptible to degradation, aggregation, and unwanted chemical modifications, leading to instability. To ensure that antibodies are suitable for production and administration, maintain stability during storage and subsequent use, and achieve optimal efficacy, research on stable formulations of antibody drugs is particularly important.

[0007] Summary of the Invention

[0008] The pharmaceutical composition described herein is a highly stable pharmaceutical composition containing an antibody that specifically binds to CD112R. The components of the pharmaceutical composition interact and synergize with each other, providing a suitable storage environment for the long-term preservation of the anti-CD112R monoclonal antibody. The composition exhibits high stability and strong tolerance to high temperatures, shaking, and repeated freeze-thaw cycles. Furthermore, the composition can block the CD112 / CD112R interaction, enhance T cell activation, mobilize the immune system, and exert a stronger tumor-killing effect.

[0009] The present invention provides a pharmaceutical composition comprising: (1) a buffer; and (2) an anti-CD112R antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 comprised by the anti-CD112R antibody or antigen-binding fragment thereof are:

[0011] LCDR1: KASQSVSNDVA (SEQ ID NO: 1);

[0012] LCDR2: YVSX1RFT, where X1 is H or N;

[0013] LCDR3: X2QAYRSPWT, where X2 is H or Q;

[0014] HCDR1: SYHMS (SEQ ID NO: 6):

[0015] HCDR2: AIX3X4X5GX6X7TYYX8DTVKG, where X3 is S or N, X4 is R or S, X5 is D or N, X6 is D or I, X7 is S or N, and X8 is P or L.

[0016] HCDR3: HEDYX9GFAMDX 10 , X9 is F or Y, X 10 It is S or Y.

[0017] In some embodiments, the anti-CD112R antibody or antigen-binding fragment thereof comprises:

[0018] LCDR1 having an amino acid sequence as shown in SEQ ID NO: 1;

[0019] LCDR2 having an amino acid sequence as shown in SEQ ID NO: 2 or 4;

[0020] LCDR3 having an amino acid sequence as shown in SEQ ID NO: 3 or 5;

[0021] HCDR1 having an amino acid sequence as shown in SEQ ID NO: 6;

[0022] HCDR2 with the amino acid sequence shown in SEQ ID NO: 7 or 9;

[0023] The HCDR3 has the amino acid sequence shown in SEQ ID NO: 8 or 10.

[0024] In some embodiments, the anti-CD112R antibody or antigen-binding fragment thereof comprises:

[0025] (1) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or

[0026] (2) LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; or

[0027] (3) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0028] In some embodiments, the anti-CD112R antibody or antigen-binding fragment thereof is selected from a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, preferably a humanized antibody or antigen-binding fragment thereof.

[0029] In some embodiments, the anti-CD112R antibody or antigen-binding fragment thereof comprises:

[0030] (1) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 12; or

[0031] (2) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 13; or

[0032] (3) a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15.

[0033] In some embodiments, the anti-CD112R antibody comprises:

[0034] (1) The light chain amino acid sequence is shown in SEQ ID NO: 16, and the heavy chain amino acid sequence is shown in SEQ ID NO: 17.

[0035] (2) The light chain amino acid sequence is shown in SEQ ID NO: 16, and the heavy chain amino acid sequence is shown in SEQ ID NO: 18.

[0036] (3) The light chain amino acid sequence is shown in SEQ ID NO: 19, and the heavy chain amino acid sequence is shown in SEQ ID NO: 20.

[0037] In some embodiments, the concentration of the anti-CD112R antibody or its antigen-binding fragment in the above-mentioned pharmaceutical composition is about 1 to 100 mg / mL, preferably about 10 to 70 mg / mL, and more preferably about 20 to 60 mg / mL; more preferably, the concentration of the above-mentioned anti-CD112R antibody or its antigen-binding fragment is about 1 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 35 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 or a range formed by any two values ​​within these ranges as endpoints, preferably about 35 mg / mL, about 40 mg / mL or about 45 mg / mL.

[0038] In some embodiments, the pH of the pharmaceutical composition is about 4.5-6.5, preferably about 5.5-6.5, more preferably about 5.5-5.7 or 5.8-6.2. Non-limiting examples of the pH of the pharmaceutical composition are about 4.5, about 5.0, 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, or a range formed by any two values ​​within these ranges as endpoints, preferably about 5.9, about 6.0 or about 6.1.

[0039] In some embodiments, the osmotic pressure of the pharmaceutical composition is about 250-350 mOsm / kg.

[0040] In some embodiments, the buffer is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is histidine buffer.

[0041] In some embodiments, the concentration of the above buffer is about 5-100 mM, preferably about 10-50 mM, preferably about 10-30 mM; preferably about 15-25 mM. Non-limiting examples of the above buffer concentration are about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 45 mM, about 50 mM or a range formed by any two values ​​within these ranges as endpoints, preferably about 15 mM, about 20 mM or about 25 mM.

[0042] In some embodiments, the pH of the buffer solution is about 4.5-6.5, preferably about 5.5-6.5, more preferably about 5.8-6.2. Non-limiting examples of the pH of the buffer solution are about 4.5, about 5.0, 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, or a range formed by any two values ​​within these ranges as endpoints, preferably about 5.9, about 6.0 or about 6.1.

[0043] In some embodiments, the buffer is a histidine buffer; preferably, the histidine buffer is a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer, preferably a histidine-histidine hydrochloride buffer. Preferably, the pH of the histidine buffer is 5.5 to 6.5, preferably 5.8 to 6.2.

[0044] In some embodiments, the above-mentioned histidine-histidine hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of about 1 to 20 mM L-histidine and about 1 to 20 mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:1 to about 1:4. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:1. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:3. In some embodiments, the histidine preparation is: a histidine buffer with a pH of about 5.5 made of about 4.5 mM L-histidine and about 15.5 mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is a histidine buffer having a pH of about 6.0 made from about 10 mM L-histidine and about 10 mM L-histidine hydrochloride. In some embodiments, the histidine preparation is a histidine buffer having a pH of about 6.5 made from about 3 mM L-histidine and about 17 mM L-histidine hydrochloride.

[0045] In some embodiments, the pH of the acetate buffer is about 4.5 to 5.7, preferably about 5.3 to 5.7. Preferably, the acetate buffer is an acetate-sodium acetate buffer. In some embodiments, the acetate-sodium acetate buffer is made up of about 1 to 20 mM acetic acid and about 1 to 20 mM sodium acetate. In some embodiments, the acetate-sodium acetate buffer is made up of acetic acid and sodium acetate in a molar ratio of about 1:2 to about 1:3. In some embodiments, the acetate-sodium acetate buffer is made up of acetic acid and sodium acetate in a molar ratio of about 1:5 to about 1:8. In some embodiments, the acetate-sodium acetate buffer is an acetate buffer made up of about 8 mM acetic acid and about 12 mM sodium acetate at a pH of about 4.5. In some embodiments, the acetate-sodium acetate buffer is an acetate buffer made up of about 6.5 mM acetic acid and about 13.5 mM sodium acetate at a pH of about 5.0. In some embodiments, the acetic acid-sodium acetate buffer is: an acetate buffer with a pH of about 5.0-6.0 made from about 2-4 mM acetic acid and about 16-18 mM sodium acetate; preferably an acetate buffer with a pH of about 5.5 made from about 3 mM acetic acid and about 17 mM sodium acetate.

[0046] In some embodiments, the buffer is a citric acid buffer. Preferably, the pH of the citric acid buffer is 5.0-6.0. Preferably, the citric acid buffer is a citric acid-sodium citrate buffer. In some embodiments, the citric acid buffer is made up of approximately 1-20 mM citric acid and approximately 1-20 mM sodium citrate. In some embodiments, the citric acid buffer is made up of citric acid and sodium citrate in a molar ratio of approximately 1:1 to 1:4. In some embodiments, the citric acid buffer is a citric acid buffer with a pH of approximately 5.0 made up of approximately 15 mM citric acid and approximately 5 mM sodium citrate. In some embodiments, the citric acid buffer is a citric acid buffer with a pH of approximately 5.5 made up of approximately 12 mM citric acid and approximately 8 mM sodium citrate. In some embodiments, the citric acid buffer is a citric acid buffer with a pH of approximately 6.0 made up of approximately 10 mM citric acid and approximately 10 mM sodium citrate.

[0047] In some embodiments, the buffer is a phosphate buffer, preferably a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. In some embodiments, the phosphate buffer is made of about 1 to 20 mM disodium hydrogen phosphate and about 1 to 20 mM sodium dihydrogen phosphate. In some embodiments, the phosphate buffer is made of disodium hydrogen phosphate and sodium dihydrogen phosphate in a molar ratio of about 1:1 to 1:4. In some embodiments, the phosphate buffer is a phosphate buffer with a pH of about 7.0 made of about 10 mM disodium hydrogen phosphate and about 10 mM sodium dihydrogen phosphate.

[0048] In some embodiments, the pharmaceutical composition further comprises a stabilizer, and the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose; preferably, the arginine salt is arginine hydrochloride. In some embodiments, the concentration of the above stabilizer is about 10-400mM, preferably about 100-300mM, preferably about 150-270mM, and preferably about 180-260mM. Non-limiting examples of the above stabilizer concentration are about 100mM, about 130mM, about 150mM, about 170mM, about 180mM, about 190mM, about 200mM, about 210mM, about 220mM, about 225mM, about 230mM, about 235mM, about 240mM, about 250mM, about 260mM or a range formed by any two values ​​within these ranges as endpoints, preferably about 190mM, about 220mM, about 230mM or about 240mM.

[0049] In some embodiments, the stabilizer is trehalose at a concentration of about 150-270 mM; or the stabilizer is sucrose at a concentration of about 150-270 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM.

[0050] In some embodiments, the stabilizer is trehalose at a concentration of about 180-260 mM, preferably about 220-240 mM, sucrose at a concentration of about 180-260 mM, preferably about 220-240 mM, a combination of sodium chloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM, or a combination of arginine hydrochloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM.

[0051] In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is mannitol at a concentration of about 100-300 mM, preferably about 150-270 mM, preferably about 180-260 mM, and non-limiting examples of the mannitol concentration are about 200 mM, about 210 mM, about 220 mM, about 225 mM, about 230 mM, about 235 mM, about 240 mM, about 250 mM, about 260 mM, or a range formed by any two values ​​within these ranges as endpoints, preferably about 225 mM, about 230 mM, or about 235 mM.

[0052] In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is sucrose at a concentration of about 100-300 mM, preferably about 150-270 mM, preferably about 180-260 mM, and non-limiting examples of the sucrose concentration are about 200 mM, about 210 mM, about 220 mM, about 225 mM, about 230 mM, about 235 mM, about 240 mM, about 250 mM, or a range formed by any two values ​​within these ranges as endpoints, preferably about 225 mM, about 230 mM, or about 235 mM.

[0053] In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose, preferably a combination of about 20-80 mM sodium chloride and about 110-170 mM sucrose, preferably a combination of about 30-70 mM sodium chloride and about 120-160 mM sucrose. Non-limiting examples of the stabilizer are a combination of about 50 mM sodium chloride and about 140 mM sucrose, or a combination of about 50 mM sodium chloride and about 150 mM sucrose.

[0054] In some embodiments, the stabilizer is a combination of arginine hydrochloride and sucrose. In some embodiments, the stabilizer is a combination of about 30-200 mM arginine hydrochloride and about 30-200 mM sucrose, preferably a combination of about 20-80 mM arginine hydrochloride and about 110-170 mM sucrose, preferably a combination of about 30-70 mM arginine hydrochloride and about 120-160 mM sucrose. Non-limiting examples of the stabilizer are a combination of about 50 mM arginine hydrochloride and about 140 mM sucrose, or a combination of about 50 mM arginine hydrochloride and about 160 mM sucrose.

[0055] In some embodiments, the pharmaceutical composition further comprises a surfactant, and the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188.

[0056] In some embodiments, the surfactant is selected from polysorbate 80.

[0057] In some embodiments, the concentration of the above surfactant is about 0.001% to 0.1%, preferably about 0.01% to 0.1%, and preferably about 0.04% to 0.06% calculated on a w / v basis; as a non-limiting example, the concentration of the above surfactant is about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.1% or a range formed by any two values ​​within these ranges as endpoints, preferably about 0.02%, about 0.04% or about 0.06%.

[0058] In some embodiments, the pharmaceutical composition comprises the components shown in any one of the following (1) to (14), or is respectively composed of the components shown in any one of the following (1) to (14):

[0059] (1) (a) about 10 to 70 mg / mL of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5, preferably about 5.5 to 6.2; (c) 150 to 270 mM trehalose; and (d) about 0.01% to 0.1% (w / v) polysorbate 80; or

[0060] (2) (a) about 10 to 70 mg / mL of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer, pH about 5.5 to 6.5, preferably about 5.5 to 6.2; (c) 150 to 270 mM sucrose; and (d) about 0.01% to 0.1% (w / v) polysorbate 80; or

[0061] (3) (a) about 10 to 70 mg / mL of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer or acetate buffer, pH of about 5.5 to 6.5, preferably about 5.5 to 6.2; (c) a stabilizer, wherein the stabilizer is a combination of arginine hydrochloride at a concentration of about 20 to 80 mM and sucrose at a concentration of about 110-170 mM; and (d) about 0.01% to 0.1% (w / v) polysorbate 80; or

[0062] (4) (a) about 10 to 70 mg / mL of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer or acetate buffer, pH of about 5.5 to 6.5, preferably about 5.5 to 6.2; (c) a stabilizer, wherein the stabilizer is a combination of sodium chloride at a concentration of about 20 to 80 mM and sucrose at a concentration of about 110-170 mM; and (d) about 0.01% to 0.1% (w / v) polysorbate 80; or

[0063] (5) (a) about 20 to 60 mg / mL of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5, preferably about 5.5 to 6.2; (c) 180 to 260 mM trehalose; and (d) about 0.04% to 0.06% (w / v) polysorbate 80; or

[0064] (6) (a) about 20 to 60 mg / mL of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5, preferably about 5.5 to 6.2; (c) 180 to 260 mM sucrose; and (d) about 0.04% to 0.06% (w / v) polysorbate 80; or

[0065] (7) (a) about 20 to 60 mg / mL of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer or acetate buffer, pH of about 5.5 to 6.5, preferably about 5.5 to 6.2; (c) a stabilizer, wherein the stabilizer is a combination of arginine hydrochloride at a concentration of about 30 to 70 mM and sucrose at a concentration of about 120-160 mM; and (d) about 0.04% to 0.06% (w / v) polysorbate 80; or

[0066] (8) (a) about 20 to 60 mg / mL of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer or acetate buffer, pH of about 5.5 to 6.5, preferably about 5.5 to 6.2; (c) a stabilizer, wherein the stabilizer is a combination of sodium chloride at a concentration of about 30 to 70 mM and sucrose at a concentration of about 120-160 mM; and (d) about 0.04% to 0.06% (w / v) polysorbate 80; or

[0067] (9) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM trehalose; and (d) about 0.02% (w / v) polysorbate 80; or

[0068] (10) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM sucrose; and (d) about 0.02% (w / v) polysorbate 80; or

[0069] (11) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM sucrose; and (d) about 0.04% (w / v) polysorbate 80; or

[0070] (12) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM sucrose; and (d) about 0.06% (w / v) polysorbate 80; or

[0071] (13) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) a stabilizer, the stabilizer being a combination of about 50 mM arginine hydrochloride and about 140 mM sucrose; and (d) about 0.02% (w / v) polysorbate 80; or

[0072] (14) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) a stabilizer, the stabilizer being a combination of sodium chloride at a concentration of about 50 mM and sucrose at a concentration of about 140 mM; and (d) about 0.02% (w / v) polysorbate 80.

[0073] In some embodiments, the pharmaceutical composition described in any of the embodiments herein is a liquid formulation or a lyophilized formulation.

[0074] In some embodiments, the pharmaceutical composition is a liquid preparation.

[0075] In some embodiments, the liquid formulation or lyophilized formulation is stable at 2-8° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

[0076] In some embodiments, the liquid formulation or lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

[0077] The present invention also provides an injection comprising the pharmaceutical composition described in any one of the schemes herein and a sodium chloride solution or a glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w / v); preferably, the concentration of the glucose solution is about 5-25% (w / v), more preferably about 5-10% (w / v).

[0078] In some embodiments, the above-mentioned pharmaceutical composition or injection is administered via intravenous injection.

[0079] The present invention also provides use of the pharmaceutical composition or injection described in any one of the schemes herein in the preparation of a medicament for treating and / or preventing a disease or condition mediated by CD112R.

[0080] The present invention also provides a pharmaceutical composition or injection as described in any one of the embodiments herein, which is used to treat and / or prevent CD112R-mediated diseases or conditions.

[0081] The present invention also provides a method for treating and / or preventing a disease or condition mediated by CD112R, comprising administering the pharmaceutical composition or injection as described in any one of the schemes herein to a subject in need thereof.

[0082] In some embodiments, the disease or condition is cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1: Binding and dissociation curves of the drug composition and recombinant human PVRIG protein.

[0084] Figure 2: Binding curves of drug compositions and negative controls with human CD112R protein.

[0085] Figure 3: Ligand competition inhibition ELISA curves of the drug composition and negative control competing with CD112 for binding to human CD112R.

[0086] FIG4 shows the binding activity of the pharmaceutical composition to CD112R on the surface of CHO hCD112R cells.

[0087] FIG5 : The pharmaceutical composition blocks the binding activity of hCD112R protein to CHO CD112 cells.

[0088] FIG6 : Activity of the pharmaceutical composition in the human CD112R / CD112 luciferase reporter gene system.

[0089] Figure 7: Tumor growth inhibitory effects of anti-CD112R humanized antibodies, anti-TIGIT antibodies and their combination. DETAILED DESCRIPTION

[0090] Definition and Description

[0091] In order to make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in this application are only for describing specific embodiments and are not intended to be limiting. All references cited herein, including patents, patent applications, papers, textbooks and the like, and the references cited therein, to the extent that they have not yet been cited, are hereby incorporated by reference in their entirety. If one or more of the incorporated documents and similar materials are different or contradictory from the present application, including but not limited to defined terms, term usage, described technology and the like, the present application shall prevail.

[0092] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes combinations of two or more polypeptides, etc.

[0093] The term "pharmaceutical composition" or "formulation" refers to a mixture containing one or more antibodies described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert its biological activity.

[0094] The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a lyophilized formulation that is weighed and suspended. The liquid formulation of the present invention is stable upon storage, and its stability does not rely on lyophilization (or other state-changing methods, such as spray drying).

[0095] The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and / or freezing to maintain stability (e.g., chemical and / or physical stability and / or biological activity).

[0096] The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (e.g., consistency, increased stability) and / or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

[0097] As used herein, "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is intended to encompass variations of ±20% or ±10% relative to the particular value, including ±5%, ±1%, and ±0.1%, as such variations are suitable for performing the disclosed methods.

[0098] The term "buffer pH of about 4.5 to 6.5" refers to an agent that, through the action of its acid / base conjugate components, renders the solution containing the agent resistant to changes in pH. The buffer used in the formulations of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.

[0099] Herein, examples of "buffers" that control the pH within this range include acetic acid, acetates (e.g., sodium acetate), succinic acid, succinates (e.g., sodium succinate), gluconic acid, histidine, histidine salts (e.g., histidine hydrochloride), methionine, citric acid (citric acid), citrate (citrate), phosphate, citrate / phosphate, imidazole, combinations thereof, and other organic acid buffers.

[0100] A "histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, for example, a histidine buffer containing histidine and histidine hydrochloride. The histidine buffer of the present invention also includes a histidine buffer containing histidine and acetate (such as sodium salt or potassium salt).

[0101] "Citrate buffer," also known as "citrate buffer," is a buffer that includes citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate buffer.

[0102] "Acetate buffer" is a buffer comprising acetate ions. Examples of acetate buffers include acetate-sodium acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like. A preferred acetate buffer is acetate-sodium acetate buffer.

[0103] "Succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, succinate-magnesium succinate, and the like. A preferred succinate buffer is succinate-sodium succinate buffer.

[0104] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include sodium dihydrogen phosphate-disodium hydrogen phosphate, potassium dihydrogen phosphate-dipotassium hydrogen phosphate, and the like. A preferred phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

[0105] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacture, storage, and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (e.g., arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, gamma-aminobutyric acid (GABA), opines, alanine, octopine, strombine, and trimethylamine N-oxide (TMAO), human serum albumin (HSA), bovine serum albumin (BSA), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAase A. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure. The stabilizer specifically used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine, glycine, and proline. Amino acids can exist in their D- and / or L- forms, but are typically L-forms. Amino acids can exist in any suitable salt, such as hydrochloride, such as arginine hydrochloride. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, and arginine hydrochloride-sucrose.

[0106] The term "surfactant" generally includes agents that protect proteins, such as antibodies, from stress induced by the air / solution interface, solution / surface induced stress, to reduce aggregation of the antibody or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronic), sodium lauryl sulfate (SDS). Herein, unless otherwise specified, the terms "concentration of polysorbate 20" and "concentration of polysorbate 80" refer to mass volume concentration (w / v), such as "about 0.02% polysorbate 80" means "0.02g of polysorbate 80 in 100mL of liquid".

[0107] The term "isotonic" means that the formulation has an osmotic pressure substantially the same as that of human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

[0108] The term "stable" formulation refers to a formulation in which the antibody substantially maintains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or upon storage. A pharmaceutical formulation may be stable even if the antibody contained therein does not retain 100% of its chemical structure or biological function after a period of storage. In some cases, an antibody formulation may be considered "stable" if it maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function after a period of storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both incorporated by reference).

[0109] The stability of the formulation can be measured by determining the percentage of native antibody remaining therein after storage at a certain temperature for a certain period of time (among other methods). Among other methods, the percentage of native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SEC-HPLC]), where "native" means not aggregated and not degraded. In some embodiments, the stability of the protein is determined as the percentage of monomeric protein in a solution with a low percentage of degradation (e.g., fragmentation) and / or aggregated protein. In some embodiments, the formulation can be stably stored at room temperature, about 25-30°C or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, with no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% aggregated form of the antibody.

[0110] Stability can be measured by determining the percentage of antibody that migrates in the more acidic fraction ("acidic form") of the main fraction of the antibody ("major charged form") during ion exchange (among other methods), where stability is inversely proportional to the percentage of the antibody in the acidic form. The percentage of "acidified" antibody can be measured by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [CEX-HPLC]), among other methods. In some embodiments, an acceptable level of stability means that after storage of the formulation at a certain temperature for a certain period of time, no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detectable in the acidic form. The storage period before stability is measured can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, the temperature at which the pharmaceutical preparation is stored can be any temperature in the range of about -80°C to about 45°C, for example, at about -80°C, about -30°C, about -20°C, about 0°C, about 2-8°C, about 5°C, about 25°C, or about 40°C.

[0111] An antibody "retains its physical stability" in the pharmaceutical composition if it shows substantially no signs of aggregation, precipitation, and / or denaturation, e.g., when visually inspected for color and / or clarity, or when measured by UV light scattering or by size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the extent that a visible precipitate forms.

[0112] The stability of a formulation, such as physical stability, can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution and is typically measured by turbidimetry and measured in nephelometric turbidity units (NTU).

[0113] The turbidity level that varies with, for example, the concentration of one or more components in the solution (e.g., protein and / or salt concentration) is also referred to as the "turbidity" or "turbid appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity. The reference standard for determining the turbidity level of a pharmaceutical composition can be based on the European Pharmacopoeia standard (European Pharmacopoeia, 4th edition, "Directorate for the Quality of Medicine of the Council of Europe" (EDQM), Strasbourg, France). According to the European Pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension having a turbidity of about 3 according to the European Pharmacopoeia standard. Turbidity measurements by turbidimetry can detect Rayleigh scattering in the absence of association or non-ideal effects, which generally varies linearly with concentration. Other methods for evaluating physical stability are well known in the art.

[0114] An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given time point is such that the antibody is considered to still retain its biological activity as defined below. Chemical stability can be assessed by, for example, detecting or quantifying chemically altered forms of the antibody. Chemical alteration can include size change (e.g., shortening), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS). Other types of chemical alteration include charge change (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

[0115] If the antibody in the pharmaceutical composition is biologically active for its intended purpose, the antibody "retains its biological activity" in the pharmaceutical composition. For example, if the anti-CD112R antibody contained in the formulation binds to CD112R with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody before storage after storage at a temperature such as 5°C, 25°C, or 45°C for a certain period of time (e.g., 1 to 12 months), the formulation of the present invention can be considered stable. Binding affinity can also be measured by, for example, ELISA or plasma resonance technology.

[0116] In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, in a pharmacological sense, refers to an amount that is effective in preventing, treating, or alleviating the symptoms of a disorder for which the antibody is effective. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of impairment or disability caused by the affliction of the disease. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by measuring the activity of the agent in in vitro assays. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of the drug that, when administered alone or in combination with other therapeutic agents to a subject at risk of the disease or a subject experiencing relapse of the disease, inhibits the progression or recurrence of the disease.

[0117] The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects / patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In specific embodiments of the invention, the subject is a human.

[0118] The terms "administer," "administer," and "treat" refer to the introduction of a composition comprising a therapeutic agent into a subject using any of the various methods or delivery systems known to those skilled in the art. Routes of administration of anti-CD112R antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to administration by injection other than enteral or topical administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intracortical, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion, as well as electroporation in vivo.

[0119] Anti-CD112R antibody

[0120] The term "antibody" as used herein should be understood to include complete antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" (or simply "antibody portion" or "antibody fragment") of an antibody as used herein refers to one or more fragments of an antibody that retains the ability to specifically bind to human CD112R or its epitope. 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 (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0121] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0122] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic 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 character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0123] The term "murine antibody" or "hybridoma antibody" as used herein refers to a monoclonal antibody against human CD112R prepared according to the knowledge and skills in the art. During preparation, a test subject is injected with the CD112R antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.

[0124] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody such as a rodent ("parent antibody"), while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0125] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein 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) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0126] The term "full-length antibody" or "intact antibody molecule" refers to an immunoglobulin molecule composed of four peptide chains: two heavy (H) chains (approximately 50-70 kDa when full-length) and two light (L) chains (approximately 25 kDa when full-length) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) separated by more conserved framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0127] The term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, designated HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently in different systems.

[0128] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997), based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and affinity propagation clustering (Affinity Propagation Clustering) based on the variability of antibody sequences. The boundaries of the CDRs of the antibodies of the present invention can be determined by a person skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0129] As used herein, "antigen-binding fragments" include fragments of antibodies or derivatives thereof, generally including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. 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 scFv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or its derivative generally retains at least 10% of the antigen-binding activity of the parent antibody. Preferably, the binding fragment or its derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody).

[0130] The anti-CD112R antibody or antigen-binding fragment thereof of the present invention includes any one of the anti-CD112R antibodies or antigen-binding fragments described in the patent application with application number CN 202110178859.1, and the entire contents disclosed in the patent application with application number CN202110178859.1 are incorporated herein by reference. In some embodiments, the CDR sequence of the antibody used in the methods and compositions of the present invention includes humanized anti-CD112R antibodies Hu16, Hu52 and Hu59 described in CN202110178859.1, and its CDR, variable region and full-length amino acid sequence are shown in Table A.

[0131] Table A: Humanized anti-CD112R antibody amino acid sequence (KABAT scheme)

[0132] In some embodiments, the anti-CD112R antibodies or antigen-binding fragments thereof used in the methods and compositions of the present invention comprise LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, respectively:

[0133] LCDR1: KASQSVSNDVA (SEQ ID NO: 1);

[0134] LCDR2: YVSX1RFT (SEQ ID NO: 21), wherein X1 is H or N;

[0135] LCDR3: X2QAYRSPWT (SEQ ID NO: 22), wherein X2 is H or Q;

[0136] HCDR1: SYHMS (SEQ ID NO: 6);

[0137] HCDR2: AIX3X4X5GX6X7TYYX8DTVKG (SEQ ID NO: 23), wherein X3 is S or N, X4 is R or S, X5 is D or N, X6 is D or I, X7 is S or N, and X8 is P or L;

[0138] HCDR3: HEDYX9GFAMDX 10 (SEQ ID NO: 24), X9 is F or Y, X 10 It is S or Y.

[0139] In some embodiments, the anti-CD112R antibody or antigen-binding fragment thereof comprises:

[0140] LCDR1 having an amino acid sequence as shown in SEQ ID NO: 1;

[0141] LCDR2 having an amino acid sequence as shown in SEQ ID NO: 2 or 4;

[0142] LCDR3 having an amino acid sequence as shown in SEQ ID NO: 3 or 5;

[0143] HCDR1 having an amino acid sequence as shown in SEQ ID NO: 6;

[0144] HCDR2 with the amino acid sequence shown in SEQ ID NO: 7 or 9;

[0145] The HCDR3 has the amino acid sequence shown in SEQ ID NO: 8 or 10.

[0146] In some embodiments, the anti-CD112R antibody or antigen-binding fragment thereof comprises:

[0147] (1) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or

[0148] (2) LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; or

[0149] (3) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0150] In some embodiments, the anti-CD112R antibody or antigen-binding fragment thereof is selected from a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, preferably a humanized antibody or antigen-binding fragment thereof.

[0151] In some embodiments, the anti-CD112R antibody or antigen-binding fragment thereof comprises:

[0152] (1) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 12; or

[0153] (2) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 13; or

[0154] (3) a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15.

[0155] In some embodiments, the anti-CD112R antibody comprises:

[0156] (1) a light chain amino acid sequence as shown in SEQ ID NO: 16, and a heavy chain amino acid sequence as shown in SEQ ID NO: 17;

[0157] (2) a light chain amino acid sequence as shown in SEQ ID NO: 16, and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; or

[0158] (3) The light chain amino acid sequence is shown in SEQ ID NO: 19, and the heavy chain amino acid sequence is shown in SEQ ID NO: 20.

[0159] In some embodiments, a non-limiting, exemplary antibody used in the Examples herein is Hu52, which is a humanized antibody comprising the light and heavy chain amino acid sequences shown in SEQ ID NOs: 16 and 18.

[0160] SEQ ID NOs: 1-20 herein are shown below:

[0161] In some embodiments, the anti-CD112R antibodies or antigen-binding fragments thereof used in the methods and compositions of the present invention are humanized antibodies or chimeric antibodies and may include a human constant region. In some embodiments, the constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions; preferably, the anti-CD112R antibodies or antigen-binding fragments thereof suitable for use in the methods and compositions of the present invention comprise a heavy chain constant region of the human IgG1 or IgG4 isotype.

[0162] In some embodiments, the present invention provides a method for preparing an anti-CD112R antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0163] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These especially include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression levels.

[0164] In one embodiment, the present invention provides a method for preparing an anti-CD112R antibody, wherein the method comprises, when an expression vector is introduced into a mammalian host cell, culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably, secreting the antibody into a culture medium in which the host cell is grown, to produce the antibody. Standard protein purification methods can be used to recover the antibody from the culture medium.

[0165] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies. Likewise, in certain embodiments, non-fucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are less likely to be immunogenic because their carbohydrate structures are normal components of natural human serum IgG.

[0166] pharmaceutical preparations

[0167] The present invention provides a pharmaceutical composition comprising: (1) a buffer; and (2) an anti-CD112R antibody or an antigen-binding fragment thereof.

[0168] The anti-CD112R antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is as described in any embodiment of the "Anti-CD112R Antibody" section of this application.

[0169] For example, the anti-CD112R antibody or its antigen-binding fragment in the pharmaceutical composition of the present invention comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10, respectively. Preferably, the above-mentioned anti-CD112R antibody or its antigen-binding fragment is selected from a murine antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, preferably a humanized antibody or its antigen-binding fragment. Preferably, the above-mentioned anti-CD112R antibody or its antigen-binding fragment comprises a light chain variable region as shown in SEQ ID NO: 11, and a heavy chain variable region as shown in SEQ ID NO: 13. More preferably, the above-mentioned anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16, and a heavy chain amino acid sequence as shown in SEQ ID NO: 18.

[0170] The concentration of the anti-CD112R antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is about 1 to 100 mg / mL, preferably about 10 to 70 mg / mL, and more preferably about 20 to 60 mg / mL. In some embodiments, the concentration of the anti-CD112R antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is 40±10 mg / mL.

[0171] The pH of the pharmaceutical composition of the present invention is about 4.5 to 6.5, preferably about 5.5 to 6.5, more preferably about 5.8 to 6.2. In some embodiments, the pH of the pharmaceutical composition of the present invention is about 5.5 or about 6.0.

[0172] The buffer in the pharmaceutical composition of the present invention is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is a histidine buffer, preferably, the histidine buffer is a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer, preferably a histidine-histidine hydrochloride buffer.

[0173] Preferably, the concentration of the buffer is about 5 to 100 mM, preferably about 10 to 50 mM, preferably about 10 to 30 mM; preferably about 15 to 25 mM. Preferably, the pH of the buffer is about 4.5 to 6.5, preferably about 5.5 to 6.5, preferably about 5.8 to 6.2. In some embodiments, the pH of the buffer of the present invention is about 5.5 or about 6.0.

[0174] Therefore, the pharmaceutical composition of the present invention may contain: a histidine buffer having a pH of about 5.5 to 6.5, whose concentration in the pharmaceutical composition is about 10 to 30 mM; and about 10 to 70 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof described in any of the foregoing embodiments, in particular the humanized antibody Hu52 or antigen-binding fragment thereof described herein.

[0175] In some embodiments, the pharmaceutical composition of the present invention further comprises a stabilizer selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose, and trehalose; preferably, the arginine salt is arginine hydrochloride. Preferably, the concentration of the stabilizer is about 10 to 400 mM, preferably about 100 to 300 mM, preferably about 150 to 270 mM, and preferably about 180 to 260 mM. It should be understood that when two or more stabilizers are used, the sum of their concentrations is also within the range of 10 to 400 mM, preferably within the range of 150 to 270 mM, and more preferably within the range of 180 to 260 mM.

[0176] Preferably, the stabilizer in the pharmaceutical composition of the present invention is: trehalose at a concentration of about 150-270 mM; or sucrose at a concentration of about 150-270 mM; or a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; or a combination of arginine hydrochloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM. In some embodiments, the stabilizer in the pharmaceutical composition of the present invention is: trehalose at a concentration of about 180-260 mM, preferably about 220-240 mM; or sucrose at a concentration of about 180-260 mM, preferably about 220-240 mM; or the above stabilizer is a combination of sodium chloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM; or the above stabilizer is a combination of arginine hydrochloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM.

[0177] Therefore, in some embodiments, the pharmaceutical composition of the present invention may contain: a histidine buffer having a pH of about 5.5 to 6.5, the concentration of which in the pharmaceutical composition is about 10 to 30 mM; about 10 to 70 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof described in any of the foregoing embodiments, in particular the humanized antibody Hu52 or antigen-binding fragment thereof described herein; and about 150 to 270 mM of a stabilizer, preferably, the stabilizer is selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose and trehalose, preferably, the arginine salt is arginine hydrochloride. Preferably, the above-mentioned stabilizer is trehalose at a concentration of about 180-260 mM, preferably about 220-240 mM; or sucrose at a concentration of about 180-260 mM, preferably about 220-240 mM; or a combination of sodium chloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM; or the above-mentioned stabilizer is a combination of arginine hydrochloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM.

[0178] In some embodiments, the pharmaceutical composition further comprises a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188. Preferably, the concentration of the surfactant is about 0.001% to 0.1%, preferably about 0.01% to 0.1%, preferably about 0.02% to 0.06%, and more preferably about 0.04% to 0.06%, calculated on a w / v basis.

[0179] Therefore, the pharmaceutical composition of the present invention may contain: a histidine buffer having a pH of about 5.5 to 6.5, the concentration of which in the pharmaceutical composition is about 10 to 30 mM; about 10 to 70 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof described in any of the above embodiments, in particular the humanized antibody Hu52 or antigen-binding fragment thereof described herein; about 150 to 270 mM of a stabilizer; and about 0.01% to 0.1% of polysorbate 80 on a w / v basis. Preferably, the stabilizer is selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose and trehalose, and preferably, the arginine salt is arginine hydrochloride. Preferably, the stabilizer is trehalose at a concentration of about 180-260 mM, preferably about 220-240 mM; or sucrose at a concentration of about 180-260 mM, preferably about 220-240 mM; or a combination of sodium chloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM; or the above stabilizer is a combination of arginine hydrochloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM.

[0180] The pharmaceutical composition of the present invention is a liquid preparation or a lyophilized preparation.

[0181] The present invention also provides an injection comprising the pharmaceutical composition described in any one of the schemes herein and a sodium chloride solution or a glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w / v); preferably, the concentration of the glucose solution is about 5-25% (w / v), more preferably about 5-10% (w / v).

[0182] In some embodiments, the above-mentioned pharmaceutical composition or injection is administered via intravenous injection.

[0183] Medical uses and methods

[0184] The present invention also provides use of the pharmaceutical composition or injection described in any one of the schemes herein in the preparation of a medicament for treating and / or preventing a disease or condition mediated by CD112R.

[0185] The present invention also provides a pharmaceutical composition or injection as described in any one of the embodiments herein for treating and / or preventing a disease or condition mediated by CD112R.

[0186] The present invention also provides a method for treating and / or preventing a disease or condition mediated by CD112R, comprising administering the pharmaceutical composition or injection as described in any one of the schemes herein to a subject in need thereof.

[0187] In the present invention, CD112R-mediated diseases refer to diseases in which CD112R participates in the occurrence and development of the disease, including but not limited to cancer.

[0188] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are both benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, nasopharyngeal cancer, esophageal squamous cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, storage diseases (bulky disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0189] The pharmaceutical composition or injection can be administered to a subject as described above, for example, by intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral administration routes, such as injection or infusion.

[0190] Example

[0191] The present invention will be described below with reference to specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The present invention has been described in detail herein, and specific embodiments thereof are disclosed therein. It will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., which are apparent to those skilled in the art without departing from the spirit and scope of the present invention, are intended to be included within the scope of the present invention. The methods and materials used in the examples are conventional methods and materials in the art, unless otherwise indicated.

[0192] Detection methods used in the examples

[0193] (1) Appearance

[0194] Inspect the appearance using a visual inspection method. Ensure the light intensity of the clarity meter is maintained between 10001x and 15001x. Hold the sample at eye level and gently shake or invert it to avoid introducing air bubbles. Perform the visual inspection against both a black and white background. Record the results for color, opalescence, and visible foreign matter.

[0195] (2) Protein content

[0196] The protein concentration was determined using a UV spectrophotometer. The percent extinction coefficient (E1%) was set at 1.389 (mg / ml). - 1 cm -1 Based on the predicted protein concentration of the test sample, dilute the sample with ultrapure water to approximately 0.4 mg / mL, maintaining an absorbance reading between 0.3 and 0.7. Each sample was assayed in duplicate, with each assay repeated three times. Before each assay, rinse the cuvette twice with 150 μl of solution, then sample 150 μl for assay.

[0197] Concentration calculation formula: C (mg / mL) = A / ε × DF; Sample protein content (mg / mL) = (C1 + C2) / 2

[0198] C is the protein content in the solution;

[0199] A is the absorbance at 280 nm, A = (A1 + A2 + A3) / 3;

[0200] ε: Extinction coefficient (1.389 (mg / mL) -1 cm -1 );

[0201] DF: dilution factor.

[0202] (3)SEC-HPLC purity

[0203] SEC-HPLC purity was determined using an HPLC (Waters e2695 instrument) equipped with a SEC column (Waters Xbrige BEH200A, 7.8*300 mm, 3.5 μm). The mobile phase consisted of 50 mM phosphate, 300 mM NaCl, pH 6.8±0.2. Peak area normalization was used for quantitative analysis. The peak area percentages of monomers, aggregates, and fragments were calculated, with the peak area percentage of monomers serving as the purity of the sample, and the peak area percentages of aggregates and fragments serving as the aggregate and fragment content. Chromatographic parameters are shown in Table 1 below.

[0204] Table 1: SEC-HPLC Chromatographic Parameters

[0205] (4) R-CE-SDS purity

[0206] Purity testing of antibody preparations by reduced CE-SDS electrophoresis uses a high-voltage DC electric field as the driving force and a capillary as the separation channel. The pre-filled gel forms a molecular sieve within the capillary. Sodium dodecyl sulfate eliminates the charge effects of different protein molecules, and the reducing agent β-mercaptoethanol cleaves disulfide bonds in the sample. Samples of varying molecular size migrate at different speeds within the capillary, thus enabling separation. The sample was diluted to 1 mg / mL with 1% SDS in 40 mM phosphate buffer, pH 6.5. Then, 95 μl was aliquoted and mixed with 5.0 μl of β-mercaptoethanol. A blank control was also prepared by diluting ultrapure water to the same dilution factor as the reference product. 95 μl was aliquoted and mixed with 5.0 μl of β-mercaptoethanol. The prepared sample was centrifuged at 3000 rpm for 30 seconds at room temperature, incubated at 70°C for 15 minutes, cooled to room temperature, and centrifuged at 12000 rpm for 5 minutes at room temperature. Separation time: 30 minutes. Detection was performed using a capillary electrophoresis instrument (Maurice, PA800). The purity of heavy chain (HC), non-glycosylated heavy chain (NGHC) and light chain (LC) was calculated, and the sum of the three was the purity of the sample.

[0207] (5) NR-CE-SDS purity

[0208] Purity testing of antibody preparations using non-reducing CE-SDS electrophoresis utilizes a high-voltage DC electric field as the driving force and a capillary as the separation channel. The pre-filled gel forms a molecular sieve within the capillary. The sample is treated with sodium dodecyl sulfate to eliminate the charge effects of different protein molecules, allowing separation of samples of varying molecular size and migration speed within the capillary. Adding an alkylating agent to the test solution effectively reduces component diffusion, resulting in sharp peaks and high separation efficiency while ensuring that the sample remains in a non-reducing state. Based on a final volume of 100 μl, calculate the sample buffer (1% SDS, 40 mM phosphate buffer, pH 6.5) and sample volume required to dilute the sample / reference product to 1 mg / mL. Then, add the buffer, followed by 5.0 μl of 0.25 M NEM, and finally the sample, to a 1.5 mL EP tube and mix thoroughly. Ultrapure water is used as a blank control, and the dilution ratio remains the same as for the reference product. The prepared samples were centrifuged at 3000 rpm at room temperature for 30 seconds, incubated at 70°C for 5 minutes, cooled to room temperature, centrifuged at 12000 rpm at room temperature for 5 minutes, and detected using a capillary electrophoresis instrument (Maurice, PA800).

[0209] (6) CEX-HPLC purity

[0210] Charge heterogeneity was determined using cation exchange chromatography (CEX-HPLC). CEX-HPLC purity was determined using an HPLC (Waters e2695 instrument) equipped with a ProPac WCX-10 column (4 x 250 mm). The mobile phase composition was phase A: 20 mM MES (pH 6.00 ± 0.02); phase B: 20 ​​mM MES + 200 mM NaCl (pH 6.00 ± 0.02). The percentages of the main peak, acidic peak, and basic peak were calculated using peak area normalization. Manual integration was used if automatic integration was not sufficient. Detailed chromatographic parameters are shown in Table 2 below.

[0211] Table 2: CEX-HPLC chromatographic parameters

[0212] (7) Binding activity

[0213] Binding ELISA: Using the indirect method, HX1 hCD112R Fc was used as the antigen to coat a 96-well plate. The antibody preparation was able to bind to HX1 hCD112R Fc. Mouse Anti-Human IgG4 Fc-HRP was able to specifically bind to the antibody preparation bound to the solid-phase antigen (HX1 hCD112R Fc). Mouse Anti-Human IgG4 Fc-HRP catalyzed TMB in the presence of hydrogen peroxide to develop a blue color. The intensity of the blue color was positively correlated with the amount of Mouse Anti-Human IgG4 Fc-HRP bound. The color turned yellow after termination with 2M hydrochloric acid. The absorbance (OD value) was measured at wavelengths of 450nm / 620nm using a microplate reader. The ability of the antibody preparation to bind to CD112R was assessed using the effective binding activity (EC50) of the standard curve.

[0214] (8) Subvisible particle (MFI) detection

[0215] The MFI Microfluidic Imaging Particle Analysis System integrates digital microscopy, microfluidics, and image processing technologies into a fully automated instrument for particle analysis. As a sample flows through the detection zone, MFI captures an image of the sample. Each particle in the image is analyzed, generating data on particle count, size, transparency, and morphology. It can also be used to analyze particle dynamics in real time. Morphological analysis software can also be used to analyze particles of specific morphologies or to isolate subpopulations of particles.

[0216] (9) iCIEF purity

[0217] iCIEF is detected using a capillary electrophoresis instrument equipped with an iCIEF cartridge. After the sample is diluted with the iCIEF mixture, it is detected using the capillary electrophoresis instrument. Components with different isoelectric points are focused at different locations, achieving both focusing and separation.

[0218] (10) Thermal stability (Tm)

[0219] When a protein is in a folded state, the emission light of its internal hydrophobic amino acids at 330nm is greater than that at 350nm. When we heat or treat the protein with chemical denaturants, the protein will unfold and when the amino acids are exposed to the liquid phase, the maximum emission light will shift, and the emission light at 350nm will eventually be greater than the emission light at 330nm. Therefore, we can use the change in fluorescence value to detect the process of protein unfolding caused by temperature or chemical denaturants by increasing the temperature from 20℃ to 95℃ at a heating rate of 1℃ / min, and thus calculate the half-melting temperature, that is, the Tm value.

[0220] (11) Aggregation temperature (Tagg)

[0221] Protein products are heated from 35°C to 85°C, with a scan rate of 1-2°C. Aggregation begins to occur to varying degrees at a certain temperature. By monitoring changes in particle diameter and temperature in the sample solution, the initial aggregation temperature (Tagg) of the protein can be determined.

[0222] Explanation of the abbreviations in the following examples: "h" represents hour, "W" represents week, "M" represents month, "C" represents the number of freeze-thaw cycles, "FT" represents freeze-thaw cycles, "SH" represents shaking, "ST" represents stirring, "RT" represents room temperature, "rpm" represents revolutions per minute, and "T0" represents the initial test of the formulation sample before the sample is subjected to the sample setting-out treatment.

[0223] Example 1: Buffer system screening experiment

[0224] In liquid pharmaceutical compositions, the buffer system closely affects the stability of the antibody. Each antibody with unique physicochemical properties has an optimal buffer type. This example aims to preliminarily screen the optimal buffer system and stabilizer to ensure that the anti-CD112R antibody disclosed in the present invention has optimal stability for clinical application.

[0225] 1.1 Experimental steps

[0226] This example was performed using antibody Hu52. The sample was prepared using Millipore Pellicon 3 0.11m 2 The sample was concentrated by UF / DF ultrafiltration to a concentration of approximately 52.5 mg / mL. The sample was then dialyzed into the corresponding formulation shown in Table 3, with the final concentration adjusted to approximately 40 mg / mL. The corresponding concentration of polysorbate 80 was then added. The sample was aseptically filled into 2R vials (2.0 mL / vial) in a clean bench for stability sampling and testing.

[0227] Table 3: Prescription information for buffer system screening experiments

[0228] 1.2 Experimental Results

[0229] 1.2.1 Appearance

[0230] According to the results in Table 4, after being placed under high temperature or long-term conditions for 4 weeks, the appearance of all samples did not show any significant changes; after being repeatedly frozen and thawed for 5 times, the appearance of all samples did not show any significant changes.

[0231] Table 4: Appearance test results

[0232] 1.2.2 SEC-HPLC purity

[0233] According to the SEC purity results in Table 5, after 4 weeks of storage at high temperature, all formulations showed an increase in aggregates and fragments, while the monomer purity of formulations FS1-1 and FS1-4 decreased significantly. Formulation FS1-8 performed relatively well. After 4 weeks of long-term storage, the SEC purity of all formulations remained unchanged. After 5 freeze-thaw cycles, the SEC purity of all samples remained unchanged.

[0234] Table 5: SEC-HPLC test results

[0235] 1.2.3 R-CE-SDS purity

[0236] According to the R-CE-SDS purity results in Table 6, after 4 weeks of storage at high temperature, the purity of formulations FS1-4 and FS1-5 decreased significantly, while the differences between the other groups were small. After 4 weeks of long-term storage and five freeze-thaw cycles, the R-CE-SDS purity of all samples did not change significantly.

[0237] Table 6: R-CE-SDS test results

[0238] 1.2.4 NR-CE-SDS purity

[0239] According to the NR-CE-SDS purity results in Table 7, after being placed under high temperature conditions for 4 weeks, the NR-CE-SDS purity of all samples decreased, among which the purity of formulations FS1-2 and FS1-4 decreased relatively quickly; after long-term storage for 4 weeks and five freeze-thaw cycles, the NR-CE-SDS purity of all samples did not change significantly.

[0240] Table 7: NR-CE-SDS test results

[0241] 1.2.5 iCIEF purity

[0242] According to the iCIEF purity results in Table 8, after 4 weeks of storage under high temperature conditions, all samples showed a significant increase in acid peaks and a decrease in the purity of the main peak. Among them, the main peak purity of formulations FS1-2, FS1-3, FS1-7, and FS1-8 decreased more slowly, and formulation FS1-4 showed the worst stability. After 4 weeks of storage under long-term conditions and five freeze-thaw cycles, the main peak purity of all samples did not change significantly.

[0243] Table 8: iCIEF test results

[0244] 1.2.6 Subvisible particles

[0245] According to the subvisible particle results in Table 9, the number of subvisible particles in formulations FS1-3, FS1-6, FS1-7, and FS1-8 increased relatively slowly after being placed under high temperature conditions for 4 weeks and subjected to 5 freeze-thaw cycles.

[0246] Table 9: Subvisible particle test results

[0247] 1.3 Buffer system screening conclusion

[0248] In summary, after four weeks of storage at high temperature, formulations FS1-3 (acetate buffer, pH 5.5) and FS1-8 (histidine buffer, pH 6.0) showed significantly higher main peak purity and fewer subvisible particles than the other formulations. Furthermore, the appearance of all formulations remained unchanged. Therefore, pharmaceutical compositions of monoclonal antibodies with 20 mM acetate buffer (pH 5.5) and 0.02% polysorbate 80(II) and 20 mM histidine buffer (pH 6.0) and 0.02% polysorbate 80(II) exhibit optimal stability at high temperature.

[0249] Example 2: Stabilizer screening experiment

[0250] To further explore the effects of different excipients on antibody stability, we conducted comparative tests using different stabilizers. We examined the effects of these different excipients on stability in a pH 5.5, 20mM acetate buffer system and a pH 6.0, 20mM histidine buffer system at a Hu52 antibody concentration of 40mg / mL.

[0251] 2.1 Experimental steps

[0252] This example was performed using antibody Hu52. The sample was prepared using Millipore Pellicon 3 0.11m 2 The sample was concentrated by UF / DF ultrafiltration to a concentration of approximately 55 mg / mL. The sample was then dialyzed into the corresponding formulation shown in Table 10, with the final concentration adjusted to approximately 40 mg / mL. The corresponding concentration of polysorbate 80 was then added. The sample was aseptically filled into 2R vials (2.0 mL / vial) in a clean bench for stability sampling and testing.

[0253] Table 10: Prescription information for stabilizer screening experiments

[0254] Note: “ / ” means none.

[0255] 2.2 Experimental Results

[0256] 2.2.1 Thermal stability (Tm)

[0257] According to the thermal stability (Tm) test results in Table 11, the Tm values ​​of formulations FS2-5 and FS2-6 were relatively high.

[0258] Table 11: Thermal stability (Tm) test results

[0259] 2.2.2 Aggregation temperature (Tagg)

[0260] According to the aggregation temperature (Tagg) test results in Table 12, the aggregation temperature of prescription FS2-5 is relatively high.

[0261] Table 12: Aggregation temperature (Tagg) test results

[0262] 2.2.3 Appearance

[0263] According to the appearance results in Table 13, all formulations had normal appearance after being placed under high temperature, accelerated or long-term conditions for 4 weeks.

[0264] Table 13: Appearance test results

[0265] 2.2.4 SEC-HPLC purity

[0266] According to the SEC-HPLC purity results in Table 14, the SEC purity of all samples decreased after being placed under high temperature, accelerated conditions or long-term conditions for 4 weeks, and there was no significant difference between the groups.

[0267] Table 14: SEC-HPLC purity test results

[0268] 2.2.5 R-CE-SDS purity

[0269] According to the R-CE-SDS purity results in Table 15, there was no significant change in sample purity after 4 weeks of storage under high temperature, accelerated and long-term conditions.

[0270] Table 15: R-CE-SDS purity test results

[0271] 2.2.6 NR-CE-SDS purity

[0272] According to the NR-CE-SDS purity results in Table 16, the purity of the samples decreased after 4 weeks under high temperature conditions, with no significant differences between the sample groups. There was no significant change in the purity of the samples after 4 weeks under accelerated and long-term conditions.

[0273] Table 16: NR-CE-SDS test results

[0274] 2.2.7 CEX-HPLC purity results

[0275] According to the CEX-HPLC purity results in Table 17, after 4 weeks of storage under high temperature conditions, the CEX-HPLC purity of all samples decreased. The rate of increase of the acidic peak in acetate buffer at pH 5.5 (FS2-1 to FS2-4) was faster than that in histidine buffer at pH 6.0 (FS2-5 to FS2-8), indicating a certain correlation between the acidic peak and pH. After 4 weeks of storage under accelerated conditions, the CEX purity of all samples decreased, but there was no significant difference between the groups. After 4 weeks of storage under long-term conditions, the CEX-HPLC purity of all samples did not change significantly.

[0276] Table 17: CEX-HPLC test results

[0277] 2.2.8 Binding activity

[0278] According to the binding activity results in Table 18, the binding activities of all samples did not show significant changes after being placed under high temperature, accelerated conditions and long-term conditions for 4 weeks.

[0279] Table 18: Binding activity test results

[0280] 2.2.9 Subvisible particles

[0281] According to the subvisible particle test results in Table 19, after being placed under high temperature, accelerated conditions or long-term conditions for 4 weeks, the subvisible particles of all samples did not show significant changes.

[0282] Table 19: Subvisible particle test results

[0283] 2.3 Stabilizer Screening Conclusion

[0284] In summary, based on the dissolution temperature results, FS2-5 (histidine buffer, pH 6.0, 230 mM sucrose) and FS2-6 (histidine buffer, pH 6.0, 230 mM trehalose) formulations were superior; based on the aggregation temperature results, FS2-5 formulation was superior. In addition, there were no significant changes in appearance, SEC purity, R-CE-SDS purity, NR-CE-SDS purity, CEX-HPLC purity, binding activity, and subvisible particles for all formulations. Therefore, the pharmaceutical composition of the monoclonal antibody with 20 mM histidine buffer (pH 6.0) and 230 mM sucrose has the best stability under high temperature conditions.

[0285] Example 3: Surfactant screening experiment

[0286] 3.1 Experimental steps

[0287] The Hu52 monoclonal antibody sample was isolated using Millipore Pellicon3 0.11m 2 Perform UF / DF ultrafiltration and concentration, exchanging approximately 8 times the volume into 20 mM histidine hydrochloride buffer (pH 6.0) containing 230 mM sucrose. Adjust the final concentration to approximately 40 mg / mL. Add the corresponding concentration of polysorbate 80 (see Table 20) for stability testing. Aseptically fill into 2R vials (2.0 mL / vial) in a clean bench for stability sampling and testing.

[0288] Table 20: Surfactant screening protocol

[0289] 3.2 Experimental Results

[0290] 3.2.1 Appearance

[0291] According to the appearance results in Table 21, there was no significant change in the appearance of all samples after being placed under high temperature, accelerated or long-term conditions for 4 weeks.

[0292] Table 21: Appearance test results

[0293] 3.2.2 SEC-HPLC purity

[0294] According to the SEC-HPLC purity results in Table 22, after 4 weeks of storage under high temperature conditions, all formulations showed a slight increase in aggregates. Under accelerated and long-term conditions, the purity of all samples did not change significantly, and no obvious differences were observed between the groups.

[0295] Table 22: SEC-HPLC purity test results

[0296] 3.2.3 R-CE-SDS purity

[0297] According to the R-CE-SDS purity results in Table 23, the purity of all formulations decreased after 4 weeks of exposure to high temperature, with no significant differences between the groups. No significant changes in the purity of any sample were observed under accelerated or long-term conditions.

[0298] Table 23: R-CE-SDS purity test results

[0299] 3.2.4 NR-CE-SDS purity

[0300] According to the NR-CE-SDS purity results in Table 24, the purity of all formulations decreased after being placed under high temperature conditions for 4 weeks. Under accelerated and long-term conditions, no significant changes were found in the purity of all samples, and no significant differences were found between the groups.

[0301] Table 24: NR-CE-SDS purity test results

[0302] 3.2.5 CEX-HPLC purity

[0303] According to the CEX-HPLC purity results in Table 25 and the CEX-HPLC purity trend chart in Figure 3-2, no significant differences were found between the groups in all purity changes.

[0304] Table 25: CEX-HPLC purity test results

[0305] 3.2.6 Binding activity

[0306] According to the binding activity results in Table 26, the binding activity of all samples did not show significant changes after being placed under high temperature, accelerated conditions and long-term conditions for 4 weeks.

[0307] Table 26: Binding activity test results

[0308] 3.2.7 Subvisible particles

[0309] According to the subvisible particle test results in Table 27, after being placed under high temperature, accelerated conditions or long-term conditions for 4 weeks, the subvisible particles of all samples did not show significant changes.

[0310] Table 27: Subvisible particle test results

[0311] 3.3 Conclusions of surfactant screening experiment

[0312] In summary, no significant differences were observed in the appearance, purity, activity, or subvisible particles of all samples under high-temperature, accelerated, and long-term conditions, demonstrating good stability across all three formulations. Therefore, the monoclonal antibody formulation with 20 mM histidine buffer (pH 6.0), 230 mM sucrose, and polysorbate 80 (0.02% to 0.06%) exhibited good stability.

[0313] Example 4: Forced degradation experiment

[0314] 4.1 Experimental steps

[0315] Samples FS3-1, FS3-2, and FS3-3 from Example 3 were used. The samples were tested for their tolerance to shaking at 200±20 rpm at room temperature for 2 and 5 days. They were also tested for their tolerance to freeze-thaw cycles at -40°C and room temperature for 3 and 5 cycles, respectively. Testing included appearance, SEC-HPLC, SDS-CE-NR / R, CEX, and subvisible particulate matter.

[0316] 4.2 Experimental Results

[0317] 4.2.1 Shaking test

[0318] According to the results in Table 28, the shaking test revealed a significant increase in opalescence and a significant increase in the number of subvisible particles in the FS3-1 formulation. Furthermore, the R-CE-SDS purity of the FS3-1 formulation decreased relatively rapidly. No significant changes were observed in the appearance, SEC-HPLC purity, R-CE-SDS purity, NR-CE-SDS purity, CEX-HPLC purity, or number of subvisible particles in the FS3-2 and FS3-3 samples.

[0319] Table 28: Shaking test results

[0320] 4.2.2 Freeze-thaw experiment

[0321] According to the results in Table 29, no significant changes were found in the appearance, SEC-HPLC purity, R-CE-SDS purity, NR-CE-SDS purity, CEX-HPLC purity, and subvisible particle count of all samples after five cycles of freezing and thawing.

[0322] Table 29: Freeze-thaw test results

[0323] 4.3 Conclusion of forced degradation experiment

[0324] After repeated freezing, thawing and shaking, the pharmaceutical composition showed no significant changes in appearance, purity, and subvisible particles of the formulation containing 20 mM histidine buffer (pH 6.0), 230 mM sucrose, and polysorbate 80 (content 0.04% to 0.06%), demonstrating that the pharmaceutical composition of the present invention has good tolerance to shaking and repeated freezing and thawing.

[0325] In summary, we investigated different buffer systems, different pH conditions, and different excipient compositions, controlled the target pH range at 5.5–6.5, and the osmotic pressure range at 250–350 mOsm / kg, and selected the optimal formulation: approximately 20 mM histidine buffer (pH approximately 6.0), approximately 230 mM sucrose, and approximately 0.04–0.06% polysorbate 80.

[0326] Example 5: Binding and Dissociation Properties of Drug Compositions to CD112R (BIACORE)

[0327] This experiment used a GE Healthcare Life Sciences Biacore T200 molecular interaction analyzer. A 40 μg / mL goat anti-human Fc fragment antibody (Jackson ImmunoResearch) was coupled to a CM5 chip surface. The drug composition (1 μg / mL, formulation number FS3-2, antibody Hu52) was then captured. A serial dilution of human PVRIG protein was then injected, and the binding signal was detected. The human PVRIG protein concentrations were adjusted to 16 nM, 8 nM, 4 nM, 2 nM, 1 nM, and 0.5 nM, with 16 nM being repeated. Kinetic model analysis was performed using Biacore T200 Evaluation Software 3.0, and affinity KD values ​​were obtained by fitting.

[0328] The experimental results are shown in Table 30 and Figure 1. The pharmaceutical composition has specific binding activity with human PVRIG protein, with a KD value of 5.77E-11M.

[0329] Table 30: Affinity results of drug composition and recombinant human PVRIG protein

[0330] Example 6: Binding activity of pharmaceutical composition (BINDING ELISA)

[0331] The experiments were performed at 37°C using a Thermo Scientific microplate reader (model: Multiskan GO). The plate was coated with 1.0 μg / mL HX1 hCD112R Fc (Suzhou Junmeng) for 90 minutes. After washing, the plate was blocked with 2% BSA for 90 minutes. A gradient dilution of the drug composition (prescription number FS3-2, antibody Hu52) and a negative control (starting at 1 μg / mL, 3-fold gradient dilution to 0.0056 ng / mL, a total of 12 concentrations) were added and incubated for 60 minutes. After washing, a 5000-fold diluted Mouse Anti-Human IgG4 Fc-HRP (Southern Biotech, 9200-05) was added as the detection antibody and incubated for 60 minutes for detection. 0.1 mg / mL TMB was then used for color development for 15 minutes. The reaction was finally terminated with 2M HCl and the plate was read at 450 nm / 620 nm. The EC values ​​were fitted using a four-parameter logistic regression (4PL) model. 50 .

[0332] The experimental results are shown in Figure 2. The pharmaceutical composition has specific binding activity with human CD112R protein. 50 The value was 1.4 ng / mL, and the negative control sample had no binding.

[0333] Example 7: Competitive Inhibition Activity of Pharmaceutical Compositions (BLOCKING ELISA)

[0334] The experiments were performed at 37°C using a Thermo Scientific microplate reader (Model: Multiskan GO). The plates were coated with 2.0 μg / mL HX1 hCD112R Fc (Suzhou Junmeng) for 90 min, washed, and blocked with 2% BSA for 90 min. The drug composition (prescription number FS3-2, antibody Hu52) and negative control (starting at 10 μg / mL, 2-fold serial dilution to 4.883 ng / mL for a total of 12 concentrations) were diluted with MX2 hPVRL2 Fc (Suzhou Junmeng) diluted to 4.0 μg / mL. The samples were added to the blocked plates and incubated for 60 min. After washing, a 5000-fold diluted Anti Mouse IgG (Fc Specific)-Peroxidase antibody produced in goat (Sigma, A2554) was added as the detection antibody and incubated for 60 min for detection. The plates were then developed with 0.1 mg / mL TMB for 15 min, and finally stained with 2M The reaction was stopped with HCl and the plate was read at 450 nm / 620 nm. A four-parameter logistic regression (4PL) model was used to fit the IC 50 .

[0335] The experimental results are shown in Figure 3. The pharmaceutical composition can specifically block the binding of human CD112R protein to CD112 protein. 50 The value was 60.0 ng / mL, and the negative control sample had no blocking effect.

[0336] Example 8: Binding of the pharmaceutical composition to cell surface CD112R

[0337] The binding of the drug composition to CD112R on the surface of CHO hCD112R cells was detected by flow cytometry. Different concentrations of drug composition (prescription number FS3-2, antibody Hu52) and control antibody (from 0.01ng / ml to 20μg / ml) were incubated with CHOhCD112R cells at 4°C for 30 minutes, and then the unbound antibodies were washed away with staining buffer. The cells were then incubated in the dark for 30 minutes with 1% Goat Anti-Human IgG4-PE fluorescent secondary antibody (v / v) prepared with staining buffer at 4°C. The cells were collected by flow cytometry and the fluorescent antibodies bound to the cell surface were detected. Finally, the raw data were analyzed with FlowJo to obtain the MFI value (mean fluorescence intensity), and the four-parameter fitting curve was performed using GraphPad Prism software to obtain the EC 50 value.

[0338] The experimental results are shown in Figure 4. The drug composition and COM701 (Compugen's anti-CD112R antibody, expressed and purified according to the sequence of US20180280506A1) have a strong affinity with CHO hCD112R cells, and the EC binding capacity of the cells is 50 The values ​​were 36.0 ng / ml and 119.2 ng / ml, respectively, and anti-KLH hIgG4 was negative, indicating that the pharmaceutical composition can specifically bind to hCD112R on the cell surface with high affinity, and the binding affinity is better than COM701.

[0339] Example 9: Pharmaceutical composition blocks the binding of HCD112R protein to cell surface CD112R

[0340] In order to detect the activity of the drug composition in competitively inhibiting the binding of CD112R protein to CD112 on the cell surface, CHO CD112 cells were incubated with a fixed concentration of hCD112R protein solution (3 μg / ml), and different concentrations of the drug composition (prescription number FS3-2, antibody Hu52) or control antibody (from 0.169 ng / ml to 30 μg / ml, 3-fold dilution) at 4 ° C for 30 minutes. The unbound antibody was then washed away with staining buffer, and the cells were incubated with 1% Goat anti-mouse IgG Fc fluorescent secondary antibody (v / v) prepared with staining buffer at 4 ° C for 30 minutes in the dark. Finally, the cells were collected by flow cytometry and the fluorescent antibodies bound to the cell surface were detected. Finally, the raw data were analyzed with FlowJo to obtain the MFI value, and the four-parameter fitting curve was performed with GraphPad Prism software to obtain the IC 50 value.

[0341] As shown in Figure 5, the drug composition and COM701 can effectively block the binding of hCD112R protein to CHO CD112 cells, while the negative control antibody has no corresponding function. 50 The activity of COM701 is 62.2 ng / ml and 84.7 ng / ml, and the activity levels of the two are comparable.

[0342] Example 10: Activity of the pharmaceutical composition in the CD112R / CD112 reporter gene assay system

[0343] This experiment used the human CD112R / CD112 luciferase reporter gene system to detect the cellular biological activity of the drug composition and control antibody COM701 (positive control) and anti-KLH-hIgG4 (negative control) in blocking the hCD112R / hCD112 inhibitory pathway and activating NFAT. First, CHO CD112 1D2 cells expressing human CD112 target cells were plated overnight in a 96-well white flat-bottom plate at 50,000 cells per well using target cell plating medium; the next day, after aspirating the medium in the cell wells, different concentrations of the drug composition (prescription number FS3-2, antibody Hu52) or control antibody (concentrations ranging from 0.15 ng / ml to 3 μg / ml, 3-fold dilution) were added to the target cells and incubated at 37°C for 30 minutes. Then, Jurkat CD112R effector cells expressing human CD112R were added to the cell plate at a density of 100,000 cells per well and incubated in a 37°C incubator for 6 hours. Finally, the ONE-Lite Luciferase Assay System was added to the cell-antibody mixture and the chemiluminescent signal was detected using a multi-function microplate reader. The EC was calculated by fitting a four-parameter regression curve using GraphPad Prism software. 50 value.

[0344] NFAT is an important transcription factor downstream of T cell receptor activation and is crucial for IL-2 secretion and T cell proliferation response. This experiment used the luciferase reporter gene method to study the ability of the drug composition to block the CD112R / CD112 signaling pathway, activate T cells and activate NFAT signaling. The experimental results are shown in Figure 6. Both the drug composition and the positive control COM701 can effectively block the CD112R / CD112 interaction, promote T cell activation, and EC 50 The values ​​were 14.2 ng / ml and 104.2 ng / ml respectively, and the activity of antibody Hu52 was significantly higher than that of the positive control.

[0345] Example 11: Pharmacodynamic evaluation of anti-CD112R antibodies, anti-TIGIT antibodies, and their combination in a human melanoma A375 mixed PBMC subcutaneous transplant tumor model

[0346] Experimental animals: 48 female NCG mice, 6-8 weeks old, weighing approximately 18-22 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0347] Anti-TIGIT antibody (JS006): Derived from the anti-TIGIT humanized antibody hu20 in PCT application number PCT / CN2020 / 101883.

[0348] The anti-CD112R antibody is the humanized antibody hu52 described herein. The anti-CD112R humanized antibody of this example was prepared according to the formula of prescription FS3-2.

[0349] A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). A375 cells in the logarithmic growth phase were collected and resuspended in HBSS (Hank's balanced salt solution) to a suitable concentration for subcutaneous tumor inoculation in NCG mice. The A375 cells used in the co-culture experiment were treated with Mitomycin C for 2 hours and then washed three times with PBS. The frozen PBMCs were taken, resuscitated and counted, and the obtained PBMCs were added to the A375 cells treated with Mitomycin C. The PBMCs were co-cultured with A375 for 5 days in RPMI 1640 medium containing IL-2 and 10% FBS. After PBMCs were co-cultured with A375 for 5 days, the PBMCs were collected and mixed with freshly digested A375 cells. PBMCs were 4×10 5 A375 cells 4×10 6 The 0.2 ml of the drug (containing 50% Matrigel) was inoculated subcutaneously on the right side of NCG mice. The day of inoculation was designated as day 0. Mice were randomly divided into groups and given medication according to their body weight. The detailed administration method, dosage and route of administration are shown in Table 31.

[0350] Table 31. Dosage regimen

[0351] Note: N: number of animals; ip: intraperitoneal injection; Q3D: once every three days. Dosage volume: Adjust the dosing volume according to the weight of the tumor-bearing mouse (0.1 mL / 10 g).

[0352] After the start of the experiment, the tumor volume and mouse body weight were measured twice a week. The experimental results are shown in Table 32 and Figure 7.

[0353] Table 32. Average tumor volume of mice in each group (Mean ± SEM)

[0354] Note: p < 0.05 is considered to be significantly different; T / C (%) is the relative tumor proliferation rate, that is, the percentage of the tumor volume of the treatment group relative to the control group at a certain time point, T and C are the tumor volumes (TV) of the treatment group and the control group at a specific time point, respectively; tumor growth inhibition rate TGI (%) = (1-T / C) × 100%.

[0355] At the end of the experiment (25 days after administration), compared with the control group, the anti-CD112R antibody hu52 at a dose level of 30 mg / kg significantly inhibited tumor growth, with a tumor growth inhibition rate of 37.68% (p < 0.05); 30 mg / kg of anti-CD112R antibody hu52 combined with 30 mg / kg of JS006 showed significant synergistic anti-tumor effect, with a tumor growth inhibition rate of 58.30% (p < 0.01).

Claims

1. A pharmaceutical composition comprising: (1) buffer; and (2) anti-CD112R antibodies or antigen-binding fragments thereof; in, The anti-CD112R antibody or antigen-binding fragment thereof comprises the following LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3: LCDR1: KASQSVSNDVA (SEQ ID NO: 1); LCDR2: YVSX1RFT, where X1 is H or N; LCDR3: X2QAYRSPWT, where X2 is H or Q; HCDR1: SYHMS (SEQ ID NO: 6); HCDR2: AIX3X4X5GX6X7TYYX8DTVKG, where X3 is S or N, X4 is R or S, X5 is D or N, X6 is D or I, X7 is S or N, and X8 is P or L. HCDR3: HEDYX9GFAMDX 10 , X9 is F or Y, X 10 is S or Y; Preferably, the concentration of the anti-CD112R antibody or antigen-binding fragment thereof is about 1 to 100 mg / mL, preferably about 10 to 70 mg / mL, more preferably about 20 to 60 mg / mL; Preferably, the pH of the pharmaceutical composition is about 4.5 to 6.5, preferably about 5.5 to 6.5; Preferably, the osmotic pressure of the pharmaceutical composition is about 250 to 350 mOsm / kg; Preferably, the pharmaceutical composition is administered by intravenous injection.

2. The pharmaceutical composition of claim 1, wherein the anti-CD112R antibody or antigen-binding fragment thereof comprises: LCDR1 having an amino acid sequence as shown in SEQ ID NO: 1; LCDR2 having an amino acid sequence as shown in SEQ ID NO: 2 or 4; LCDR3 having an amino acid sequence as shown in SEQ ID NO: 3 or 5; HCDR1 having an amino acid sequence as shown in SEQ ID NO: 6; HCDR2 with the amino acid sequence shown in SEQ ID NO: 7 or 9; HCDR3 having an amino acid sequence as shown in SEQ ID NO: 8 or 10; Preferably, the anti-CD112R antibody or antigen-binding fragment thereof comprises: (1) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or (2) LCDR1, whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. LCDR2 and LCDR3, and HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10, respectively; or (3) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are set forth in SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and HCDR1, HCDR2, and HCDR3 whose amino acid sequences are set forth in SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; Preferably, the anti-CD112R antibody or antigen-binding fragment thereof is selected from a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, preferably a humanized antibody or antigen-binding fragment thereof.

3. The pharmaceutical composition of claim 1 or 2, wherein the anti-CD112R antibody or antigen-binding fragment thereof comprises: (1) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 12; or (2) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 13; or (3) a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15; Preferably, the anti-CD112R antibody comprises: (1) a light chain amino acid sequence as shown in SEQ ID NO: 16, and a heavy chain amino acid sequence as shown in SEQ ID NO: 17; (2) a light chain amino acid sequence as shown in SEQ ID NO: 16, and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (3) The light chain amino acid sequence is shown in SEQ ID NO: 19, and the heavy chain amino acid sequence is shown in SEQ ID NO:

20.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the buffer is selected from one or more of an acetate buffer, a citrate buffer, a phosphate buffer, and a histidine buffer; preferably, the buffer is a histidine buffer, preferably, the histidine buffer is a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer, preferably a histidine-histidine hydrochloride buffer; preferably, the concentration of the buffer is about 5 to 100 mM, preferably about 10 to 50 mM, preferably about 10 to 30 mM; preferably, the pH of the buffer is about 4.5 to 6.5, preferably about 5.5 to 6.

5.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition further comprises a stabilizer selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose, and trehalose; preferably, the arginine salt is arginine hydrochloride; preferably, the concentration of the stabilizer is about 100 to 300 mM, preferably about 150 to 270 mM, and more preferably about 180 to 260 mM.

6. The pharmaceutical composition according to claim 5, wherein The stabilizer is trehalose at a concentration of about 150-270 mM, sucrose at a concentration of about 150-270 mM, a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM, or a combination of arginine hydrochloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; Preferably, the stabilizer is trehalose at a concentration of about 180-260 mM, preferably about 220-240 mM, sucrose at a concentration of about 180-260 mM, preferably about 220-240 mM, a combination of sodium chloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM, or a combination of arginine hydrochloride at a concentration of about 30-70 mM, preferably about 40-60 mM, and sucrose at a concentration of about 120-160 mM, preferably about 130-150 mM.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition further comprises a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188; preferably, the concentration of the surfactant is about 0.01% to 0.1%, preferably about 0.02% to 0.06%, and more preferably about 0.04% to 0.06%, calculated on a w / v basis.

8. The pharmaceutical composition according to any one of claims 1 to 7, comprising the components shown in any one of the following (1) to (14): (1) (a) about 10 to 70 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5; (c) 150 to 270 mM trehalose; and (d) about 0.01% to 0.1% (w / v) polysorbate 80; or (2) (a) about 10 to 70 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5; (c) 150 to 270 mM sucrose; and (d) about 0.01% to 0.1% (w / v) polysorbate 80; or (3) (a) about 10 to 70 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5; (c) a stabilizer, the stabilizer being a combination of about 20 to 80 mM arginine hydrochloride and about 110 to 170 mM sucrose; and (d) about 0.01% to 0.1% (w / v) polysorbate 80; or (4) (a) about 10 to 70 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5; (c) a stabilizer, wherein the stabilizer is a combination of about 20 to 80 mM sodium chloride and about 110 to 170 mM sucrose; and (d) about 0.01% to 0.1% (w / v) polysorbate 80; or (5) (a) about 20 to 60 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5; (c) 180 to 260 mM trehalose; and (d) about 0.04% to 0.06% (w / v) polysorbate 80; or (6) (a) about 20 to 60 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5; (c) 180 to 260 mM sucrose; and (d) about 0.04% to 0.06% (w / v) polysorbate 80; or (7) (a) about 20 to 60 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5; (c) a stabilizer, the stabilizer being a combination of about 30 to 70 mM arginine hydrochloride and about 120 to 160 mM sucrose; and (d) about 0.04% to 0.06% (w / v) polysorbate 80; or (8) (a) about 20 to 60 mg / mL of the anti-CD112R antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer or acetate buffer, pH about 5.5 to 6.5; (c) a stabilizer, wherein the stabilizer is a combination of about 30 to 70 mM sodium chloride and about 120 to 160 mM sucrose; and (d) about 0.04% to 0.06% (w / v) polysorbate 80; or (9) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM trehalose; and (d) about 0.02% (w / v) polysorbate 80; or (10) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM sucrose; and (d) about 0.02% (w / v) polysorbate 80; or (11) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM sucrose; and (d) about 0.04% (w / v) polysorbate 80; or (12) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM sucrose; and (d) about 0.06% (w / v) polysorbate 80; or (13) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) a stabilizer, the stabilizer being a combination of about 50 mM arginine hydrochloride and about 140 mM sucrose; and (d) about 0.02% (w / v) polysorbate 80; or (14) (a) about 40 mg / mL of an anti-CD112R antibody, preferably, the anti-CD112R antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain amino acid sequence as shown in SEQ ID NO: 18; (b) about 20 mM histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) a stabilizer, the stabilizer being a combination of sodium chloride at a concentration of about 50 mM and sucrose at a concentration of about 140 mM; and (d) about 0.02% (w / v) polysorbate 80.

9. An injection comprising the pharmaceutical composition according to any one of claims 1 to 8 and a sodium chloride solution or a glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w / v); preferably, the concentration of the glucose solution is about 5-25% (w / v), preferably about 5-10% (w / v).

10. Use of the pharmaceutical composition according to any one of claims 1 to 8 or the injection according to claim 9 in the preparation of a medicament for treating and / or preventing a disease or condition mediated by CD112R; preferably, the disease or condition is cancer.

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