Anti-pd-1 antibody pharmaceutical compositions and uses thereof

Abstract

The present disclosure relates to anti-PD-1 antibody pharmaceutical compositions and uses thereof. In particular, the present disclosure provides a pharmaceutical composition comprising an anti-PD-1 antibody and a buffer.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of pharmaceutical preparations, in particular to pharmaceutical compositions comprising anti-PD-1 antibodies, and their use as medicaments. BACKGROUND

[0002] The statements herein are provided only to enhance understanding of the present disclosure and do not necessarily constitute prior art.

[0003] Tumor immunotherapy is a treatment method that takes full advantage of and mobilizes the killing T cells in the body of a tumor patient to kill the tumor. At the same time, tumor cell escape is a great obstacle faced by tumor immunotherapy, and tumor cells promote the rapid growth of tumors by using their own inhibition of the immune system. There is a very complex relationship between the immune escape mechanism of the tumor and the immune response of the body to the tumor. In the early stage of tumor immunotherapy, tumor-specific killer T cells are biologically active, but lose the function of killing in the later stage as the tumor grows.

[0004] The activation of T cells in the human body takes two signal pathway systems. In addition to the first signal provided by the antigen-presenting cells to the T cells by presenting MHC-antigen peptides, a series of costimulatory molecules also provide the second signal, so that the T cells can produce a normal immune response. This double signal pathway system plays a crucial role in the balance of the immune system in the body, and it strictly regulates the body to produce different immune responses to self and non-self antigens. If the second signal provided by the costimulatory molecules is lacking, it will lead to no response or sustained specific immune response of the T cells, thereby producing tolerance. Therefore, the second signal pathway plays a very key regulatory role in the whole process of the immune response of the body.

[0005] Programmed death-l (PD-l) is a protein receptor expressed on the surface of T cells discovered in 1992, which is involved in the process of apoptosis. PD-l belongs to the CD28 family and has 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (CTLA-4), but its expression is different from CTLA, mainly expressed on activated T cells, B cells and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed on T cells, B cells, macrophages and dendritic cells (DC), and the expression on the activated cells can be up-regulated. The expression of PD-L2 is relatively limited, mainly expressed on antigen-presenting cells such as activated macrophages and dendritic cells.

[0006] The anti-PD-1 antibody can maximize the patient's own immune system response to the tumor by blocking the binding between PD-L1 / PD-1, so as to achieve the purpose of killing tumor cells. SUMMARY

[0007] The present disclosure provides anti-PD-1 antibody pharmaceutical compositions and uses thereof.

[0008] In one aspect, the present disclosure provides a pharmaceutical composition comprising an anti-PD-1 antibody and a buffer, wherein the buffer is acetate buffer, histidine buffer or succinate buffer. Preferably, the pH of the buffer is about 4.5 to about 6.0, preferably the pH is about 4.7 to about 5.7, more preferably the pH is about 5.2; the acetate buffer is preferably acetic acid-sodium acetate buffer, the histidine buffer is preferably histidine-acetate buffer, and the succinate buffer is preferably succinic acid-sodium succinate buffer; the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 65, HCDR2 as shown in SEQ ID NO: 66, and HCDR3 as shown in SEQ ID NO: 67, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 68, LCDR2 as shown in SEQ ID NO: 12, and LCDR3 as shown in SEQ ID NO: 69; preferably, wherein the heavy chain variable region of the anti-PD-1 antibody comprises HCDR1 as shown in SEQ ID NO: 8, HCDR2 as shown in SEQ ID NO: 9, and HCDR3 as shown in SEQ ID NO: 10, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 49, LCDR2 as shown in SEQ ID NO: 12, and LCDR3 as shown in SEQ ID NO: 13. The specific sequences are shown in Table 1 below:

[0009] Table 1. Antibody CDR sequences

[0010]

[0011] In some alternative embodiments, in the aforementioned pharmaceutical composition, the heavy chain variable region of the anti-PD-1 antibody comprises HCDR1 as shown in SEQ ID NO: 8, HCDR2 as shown in SEQ ID NO: 9, and HCDR3 as shown in SEQ ID NO: 10; and the light chain variable region comprises LCDR2 as shown in SEQ ID NO: 12, LCDR3 as shown in SEQ ID NO: 13, and LCDR1 as shown in general formula RSSQSX 13 VHSX 14 X15 X 16 LCDR1 as set forth in SEQ ID NO: 68, wherein X 13 selected from L, X 14 selected from N, Q, L, T or D, X 15 selected from G, A or V, X 16 selected from N.

[0012] In some alternative embodiments, in the aforementioned pharmaceutical composition, the anti-PD-1 antibody is an anti-PD-1 antibody selected from any one of (a) to (e) as follows:

[0013] (a) an anti-PD-1 antibody comprising HCDR1, HCDR2 and HCDR3 of sequences as set forth in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and LCDR2 and LCDR3 of sequences as set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and LCDR1 of sequence as set forth in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52;

[0014] (b) an anti-PD-1 antibody comprising HCDR1, HCDR2 and HCDR3 of sequences as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and LCDR1, LCDR2 and LCDR3 of sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 18, respectively;

[0015] (c) an anti-PD-1 antibody comprising HCDR1, HCDR2 and HCDR3 of sequences as set forth in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively, and LCDR1, LCDR2 and LCDR3 of sequences as set forth in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

[0016] (d) an anti-PD-1 antibody comprising HCDR1, HCDR2 and HCDR3 of sequences as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and LCDR2 and LCDR3 of sequences as set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and LCDR1 of sequence as set forth in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52; and

[0017] (e) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 17, SEQ ID NO: 12, and SEQ ID NO: 18, respectively.

[0018] In some embodiments, in the foregoing pharmaceutical compositions, the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 49, SEQ ID NO: 12, and SEQ ID NO: 13, respectively.

[0019] In some embodiments, in the foregoing pharmaceutical compositions, the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

[0020] In some embodiments, in the foregoing pharmaceutical compositions of the disclosure, the anti-PD-1 antibody has a dissociation equilibrium constant (KD value) of equal to or less than 10 -7 In some embodiments, the anti-PD-1 antibody has a dissociation equilibrium constant (KD value) of equal to or less than 10 -8 In some embodiments, the anti-PD-1 antibody has a dissociation equilibrium constant (KD value) of equal to or less than 10 -9 In some embodiments, the anti-PD-1 antibody has a dissociation equilibrium constant (KD value) of equal to or less than 10 -10 In some embodiments, the anti-PD-1 antibody has a dissociation equilibrium constant (KD value) of equal to or less than 10 -11 In some embodiments, the anti-PD-1 antibody has a dissociation equilibrium constant (KD value) of equal to or less than 10

[0021] In some embodiments, in the foregoing pharmaceutical compositions, the pH of the buffer is about 4.5 to about 6.0, preferably 4.5 to 6.0; in some embodiments, the pH of the buffer is about 4.7 to about 5.7, preferably 4.7 to 5.7; in other embodiments, the pH of the buffer is about 5.2, preferably 5.2; in other embodiments, the buffer is an acetate buffer having a pH of about 4.7 to about 5.7; in other embodiments, the buffer is an acetate buffer having a pH of 5.2. In other embodiments, non-limiting examples of the pH of the buffer include about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, and any range between these point values. In some embodiments, the pH of the foregoing pharmaceutical compositions is consistent or nearly consistent with the pH of its buffer (as known to those skilled in the art, there can be a pH drift (some difference between the pH of the formulation and the pH of the buffer, referred to as the pH drift value) during the process of preparing a pharmaceutical formulation, and the pH drift value is typically within ±0.3, and unless otherwise specified, the pH drift value of the pharmaceutical compositions of the present disclosure is within ±0.3, preferably within ±0.2, and more preferably within ±0.1).

[0022] In some embodiments, in the foregoing pharmaceutical compositions, the concentration of the buffer is about 5 mM to about 30 mM, preferably 5 mM to 30 mM; in some embodiments, the concentration of the buffer is about 5 mM to about 15 mM, preferably 5 mM to 15 mM; in some embodiments, the concentration of the buffer is about 10 mM, preferably 10 mM. Non-limiting examples of the concentration of the buffer include about 5 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 18 mM, about 20 mM, about 25 mM, about 30 mM, and any range between these point values.

[0023] In some embodiments, in the foregoing pharmaceutical compositions, the anti-PD-1 antibody concentration is about 1 mg / mL to about 150 mg / mL, preferably 1 mg / mL to 150 mg / mL; in some embodiments, the anti-PD-1 antibody concentration is about 90 mg / mL to about 150 mg / mL, preferably 90 mg / mL to 150 mg / mL; in some embodiments, the anti-PD-1 antibody concentration is about 100 mg / mL to about 120 mg / mL. In some embodiments, the anti-PD-1 antibody concentration is about 100 mg / mL. In some embodiments, the anti-PD-1 antibody concentration is 100 mg / mL. Non-limiting examples of anti-PD-1 antibody concentrations include: about 1 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, and any range between these point values.

[0024] In some embodiments, the aforementioned pharmaceutical composition further comprises an osmotic pressure modifier. In some embodiments, the osmotic pressure modifier is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like), an amino acid (including arginine, glycine, cysteine, histidine, and the like), or a salt (sodium chloride, potassium chloride, calcium chloride, and the like). In some embodiments, the sugar is selected from the group consisting of: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol (also known as sorbose), mannitol, melibiose, raffinose, melezitose, maltotriose, stachyose, maltulose, lactulose, isomaltulose, lactitol, and isomalt. In some embodiments, the sugar is a non-reducing disaccharide; in some embodiments, the sugar is preferably trehalose or sucrose, most preferably sucrose. In some embodiments, the osmotic pressure modifier is selected from one or more of the group consisting of sucrose, trehalose, sorbitol, arginine, glycine, and sodium chloride. In some embodiments, the osmotic pressure modifier concentration is preferably about 50 mg / mL to about 100 mg / mL, more preferably about 70 mg / mL to about 90 mg / mL; most preferably about 80 mg / mL. In some embodiments, non-limiting examples of osmotic pressure modifier concentrations include about 50 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, and any range between these point values. In some embodiments, the aforementioned pharmaceutical composition is an isotonic formulation. In some embodiments, the osmotic pressure modifier controls the osmotic pressure of the aforementioned pharmaceutical composition to be between 280-320 mOsm, preferably about 300 mOsm. In some embodiments, the osmotic pressure modifier is preferably about 70 mg / mL to about 90 mg / mL sucrose, most preferably about 80 mg / mL sucrose.

[0025] In some embodiments, the foregoing pharmaceutical composition further comprises a surfactant, which can be selected from the group consisting of polysorbate 20 (also known as Tween 20), polysorbate 80 (also known as Tween 80), polyoxyl, Triton, sodium lauryl sulfate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl- sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl- sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauryl- amidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl- betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl- dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyltaurate, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol, and the like. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0026] In some embodiments, the concentration of the surfactant in the foregoing pharmaceutical composition is about 0.1 mg / mL to about 1.0 mg / mL, preferably about 0.2 mg / mL to about 0.8 mg / mL; in some embodiments, the concentration of the surfactant is about 0.4 mg / mL to about 0.8 mg / mL, preferably about 0.6 mg / mL to about 0.8 mg / mL; in some embodiments, the concentration of the surfactant is about 0.6 mg / mL, preferably 0.6 mg / mL. Non-limiting examples include about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.45 mg / mL, about 0.5 mg / mL, about 0.55 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, and any range between these point values.

[0027] In some embodiments, the anti-PD-1 antibody in the foregoing pharmaceutical composition is a murine antibody, a chimeric antibody, a fully human antibody, or a humanized antibody.

[0028] In some embodiments, the anti-PD-1 antibody in the aforementioned pharmaceutical composition is a humanized antibody. In some embodiments, the humanized antibody comprises a framework region derived from a human antibody or a framework region variant thereof. In some embodiments, the framework region variant is a back-mutation of up to 11 amino acids in the light chain framework region and / or the heavy chain framework region of a human antibody. In some embodiments, the framework region variant comprises a mutation selected from any one of (f) to (h) as described below, as compared to the framework region derived from a human antibody:

[0029] (f) comprising a 2G amino acid back-mutation in the light chain variable region, and / or comprising one or more amino acid back-mutations selected from 27Y, 48I, 67T, 69L, 82F and 93T in the heavy chain variable region;

[0030] (g) comprising a 2V amino acid back-mutation in the light chain variable region, and / or comprising one or more amino acid back-mutations selected from 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F and 93T in the heavy chain variable region; and

[0031] (h) comprising one or more amino acid back-mutations selected from 42G, 44V and 71Y in the light chain variable region, and / or comprising a 1K and / or 94S amino acid back-mutation in the heavy chain variable region.

[0032] In some embodiments, the anti-PD-1 antibody in the aforementioned pharmaceutical composition comprises an antibody variable region selected from the following:

[0033] (a2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and a heavy chain framework region comprising one or more amino acid back-mutations selected from 27Y, 48I, 67T, 69L, 82F and 93T, and

[0034] a light chain variable region comprising LCDR2 and LCDR3 as set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and LCDR1 as set forth in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52, and a light chain framework region comprising a 2G amino acid back-mutation;

[0035] (b2) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, and a heavy chain framework region comprising one or more amino acid back mutations selected from 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F, and 93T; and

[0036] a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 12, and SEQ ID NO: 18, respectively, and a light chain framework region comprising a 2V amino acid back mutation;

[0037] (c2) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and a heavy chain framework region comprising a 1K and / or 94S amino acid back mutation, and

[0038] a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively, and a light chain framework region comprising one or more amino acid back mutations selected from 42G, 44V, and 71Y.

[0039] In some embodiments, the anti-PD-1 antibody in the aforementioned pharmaceutical composition comprises an antibody variable region as set forth in any one of (i) to (o) below;

[0040] (i) a heavy chain variable region comprising a sequence as set forth in SEQ ID NO: 4 or having at least 90% sequence identity to SEQ ID NO: 4, and / or

[0041] a light chain variable region comprising a sequence as set forth in SEQ ID NO: 5 or having at least 90% sequence identity to SEQ ID NO: 5;

[0042] (j) a heavy chain variable region comprising a sequence as set forth in SEQ ID NO: 6 or having at least 90% sequence identity to SEQ ID NO: 6, and / or

[0043] a light chain variable region comprising a sequence as set forth in SEQ ID NO: 7 or having at least 90% sequence identity to SEQ ID NO: 7;

[0044] (k) a heavy chain variable region comprising a sequence as set forth in SEQ ID NO: 19 or having at least 90% sequence identity to SEQ ID NO: 19, and / or

[0045] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0046] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0047] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0048] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0049] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0050] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0051] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0052] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0053] a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0054] (p) a heavy chain variable region having a sequence of SEQ ID NO: 27, 30, 31, or 32, or at least 90% sequence identity to SEQ ID NO: 27, 30, 31, or 32, and / or

[0055] a light chain variable region having a sequence of SEQ ID NO: 34 or 35, or at least 90% sequence identity to SEQ ID NO: 34 or 35;

[0056] wherein the sequences of SEQ ID NO: 70 and SEQ ID NO: 71 are general formulae, and the specific sequences are shown in Table 2:

[0057] Table 2. Antibody variable region sequences

[0058]

[0059] In some embodiments, in the foregoing pharmaceutical composition, the heavy chain variable region of the anti-PD-1 antibody has a sequence of SEQ ID NO: 27 or at least 90% identity to SEQ ID NO: 27, and the light chain variable region of the anti-PD-1 antibody has a sequence of SEQ ID NO: 55 or at least 90% sequence identity to SEQ ID NO: 55.

[0060] In some embodiments, in the foregoing pharmaceutical composition, the heavy chain variable region of the anti-PD-1 antibody has a sequence of SEQ ID NO: 46 or at least 90% identity to SEQ ID NO: 46, and the light chain variable region of the anti-PD-1 antibody has a sequence of SEQ ID NO: 43 or at least 90% sequence identity to SEQ ID NO: 43.

[0061] The foregoing "at least 90% identity" includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0062] In some embodiments, the anti-PD-1 antibody comprises a heavy chain constant region selected from the group consisting of human IgGl, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, and a light chain constant region selected from the group consisting of human antibody kappa and lambda chain constant regions and conventional variants thereof; in some embodiments, the heavy chain constant region comprises an IgG4 heavy chain constant region incorporating one or more mutations of S228P, F234A, and L235A, e.g., three amino acid mutations of S228P, F234A, and L235A; in some embodiments, the antibody comprises a heavy chain constant region having the sequence set forth in SEQ ID NO: 72 or as set forth in SEQ ID NO: 79, and a light chain constant region having the sequence set forth in SEQ ID NO: 73.

[0063] In some embodiments, the anti-PD-1 antibody comprises a heavy chain having the sequence set forth in SEQ ID NO: 74 or 77, or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 74 or 77, and a light chain having the sequence set forth in SEQ ID NO: 75 or 78, or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 75 or 78.

[0064] In some embodiments, the anti-PD-1 antibody comprises a heavy chain having the sequence set forth in SEQ ID NO: 74 or 77, or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 74 or 77, and a light chain having the sequence set forth in SEQ ID NO: 75 or 78, or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 75 or 78.

[0065] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0066] a) an anti-PD-1 antibody at a concentration of about 1 mg / mL to about 150 mg / mL,

[0067] b) an acetate buffer at a concentration of about 5 mM to about 30 mM, at a pH of about 4.5 to about 6.0,

[0068] c) an osmotic adjusting agent at a concentration of about 50 mg / mL to about 100 mg / mL, the osmotic adjusting agent selected from the group consisting of sucrose, trehalose, sorbitol, arginine, glycine, and sodium chloride;

[0069] d) a polysorbate at a concentration of about 0.2 mg / mL to about 0.8 mg / mL;

[0070] In some embodiments, the foregoing pharmaceutical composition comprises:

[0071] A1) an anti-PD-1 antibody at a concentration of about 90 mg / mL to about 150 mg / mL,

[0072] B1) an acetate buffer at a concentration of about 10 mM to about 30 mM, at a pH of about 4.7 to about 5.7,

[0073] C1) sucrose at a concentration of about 70 mg / mL to about 90 mg / mL, and

[0074] D1) polysorbate 80 at a concentration of about 0.6 mg / mL to about 0.8 mg / mL;

[0075] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 120 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0076] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM acetate-sodium acetate buffer at a pH of about 5.0, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0077] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0078] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of acetate-sodium acetate buffer at a pH of about 5.5, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0079] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of acetate-sodium acetate buffer at a pH of about 5.7, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0080] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of histidine-acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0081] In some embodiments, the foregoing pharmaceutical composition comprises: about 30 mM of acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 120 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0082] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.2 mg / mL.

[0083] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.4 mg / mL.

[0084] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.8 mg / mL.

[0085] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 20 at a concentration of about 0.6 mg / mL.

[0086] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, trehalose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0087] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0088] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, sorbitol at a concentration of about 50 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0089] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, arginine at a concentration of about 100 mM, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0090] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, glycine at a concentration of about 100 mM, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0091] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetate-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg / mL, NaCl at a concentration of about 100 mM, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0092] In some embodiments, the foregoing pharmaceutical composition comprises: about 20 mM of an acetate-sodium acetate buffer at a pH of about 5.7, an anti-PD-1 antibody at a concentration of about 150 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0093] In some embodiments, the foregoing pharmaceutical composition comprises: about 21.9 mM of an acetic acid-sodium acetate buffer at about pH 4.7, an anti-PD-1 antibody at a concentration of about 120 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0094] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetic acid-sodium acetate buffer at about pH 4.7, an anti-PD-1 antibody at a concentration of about 90 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0095] In some embodiments, the foregoing pharmaceutical composition comprises: about 20 mM of an acetic acid-sodium acetate buffer at about pH 5.2, an anti-PD-1 antibody at a concentration of about 120 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0096] In some embodiments, the foregoing pharmaceutical composition comprises: about 20 mM of an acetic acid-sodium acetate buffer at about pH 5.2, an anti-PD-1 antibody at a concentration of about 90 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0097] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetic acid-sodium acetate buffer at about pH 5.7, an anti-PD-1 antibody at a concentration of about 150 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0098] In some embodiments, the foregoing pharmaceutical composition comprises: about 30 mM of an acetic acid-sodium acetate buffer at about pH 4.7, an anti-PD-1 antibody at a concentration of about 90 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0099] In some embodiments, the foregoing pharmaceutical composition comprises: about 10 mM of an acetic acid-sodium acetate buffer at about pH 4.7, an anti-PD-1 antibody at a concentration of about 150 mg / mL, sucrose at a concentration of about 80 mg / mL, and polysorbate 80 at a concentration of about 0.6 mg / mL.

[0100] In some embodiments, the aforementioned pharmaceutical composition comprises: approximately 30 mM acetic acid-sodium acetate buffer with a pH of approximately 5.2, an anti-PD-1 antibody at a concentration of approximately 150 mg / mL, sucrose at a concentration of approximately 80 mg / mL, and polysorbate 80 at a concentration of approximately 0.6 mg / mL.

[0101] In some embodiments, the aforementioned pharmaceutical composition comprises: approximately 10 mM acetic acid-sodium acetate buffer having a pH of approximately 5.7, an anti-PD-1 antibody at a concentration of approximately 90 mg / mL, sucrose at a concentration of approximately 80 mg / mL, and polysorbate 80 at a concentration of approximately 0.6 mg / mL.

[0102] In some embodiments, the aforementioned pharmaceutical composition comprises: approximately 30 mM acetic acid-sodium acetate buffer having a pH of approximately 5.7, an anti-PD-1 antibody at a concentration of approximately 107.7 mg / mL, sucrose at a concentration of approximately 80 mg / mL, and polysorbate 80 at a concentration of approximately 0.6 mg / mL.

[0103] In some embodiments, the aforementioned pharmaceutical composition comprises: 10 mM acetic acid-sodium acetate buffer with a pH of 5.2, an anti-PD-1 antibody at a concentration of 120 mg / mL, sucrose at a concentration of 70 mg / mL, and polysorbate 80 at a concentration of 0.6 mg / mL.

[0104] In some embodiments, the aforementioned pharmaceutical composition comprises: 10 mM acetic acid-sodium acetate buffer with a pH of 5.2, an anti-PD-1 antibody at a concentration of 120 mg / mL, sucrose at a concentration of 90 mg / mL, and polysorbate 80 at a concentration of 0.6 mg / mL.

[0105] In some embodiments, the aforementioned pharmaceutical composition comprises: a 10 mM acetic acid-sodium acetate buffer at a pH of 5.2, an anti-PD-1 antibody at a concentration of 120 mg / mL, sucrose at a concentration of 80 mg / mL, and polysorbate 80 at a concentration of 0.6 mg / mL; wherein the anti-PD-1 antibody has a heavy chain as shown in SEQ ID: 74 and a light chain as shown in SEQ ID: 75. In some embodiments, the present disclosure provides a method for preparing the aforementioned pharmaceutical composition, the method comprising the step of exchanging a stock solution of the anti-PD-1 antibody with a buffer, wherein in some embodiments, the buffer is selected from acetate buffer, histidine buffer, or succinate buffer.

[0106] The present disclosure also provides a lyophilized preparation, which can form any of the aforementioned pharmaceutical compositions after reconstitution.

[0107] In some embodiments, the present disclosure provides a lyophilized formulation comprising an anti-PD-1 antibody, which is obtained by lyophilizing the aforementioned pharmaceutical composition. In alternative embodiments, the aforementioned lyophilized formulation comprises the steps of pre-freezing, primary drying and secondary drying, in sequence.

[0108] In some embodiments, the present disclosure provides a lyophilized formulation comprising an anti-PD-1 antibody, which is reconstituted to form the aforementioned pharmaceutical composition.

[0109] In some embodiments, the present disclosure provides a lyophilized formulation, which is a lyophilized form of the aforementioned pharmaceutical composition.

[0110] In some embodiments, the aforementioned pharmaceutical composition or lyophilized formulation is a stable formulation, in some embodiments, the aforementioned pharmaceutical composition or lyophilized formulation has a decrease in SEC monomer percentage of no more than 5% when stored at refrigerated temperature (2-8°C) for 6 months. In some embodiments, the aforementioned pharmaceutical composition 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. In some embodiments, the aforementioned pharmaceutical composition has a decrease in SEC monomer percentage of no more than 2% (e.g., less than 2%, less than 1%, less than 0.8%, less than 0.5%, or even less) when stored at 4°C for 6 months; in some embodiments, the aforementioned pharmaceutical composition has a decrease in SEC of less than or equal to about 10% (e.g., less than or equal to about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%) from the SEC value at DO when stored under forced degradation conditions (40°C Ml); preferably, the decrease in SEC is between 1.1% and 2.7%. In some embodiments, the aforementioned pharmaceutical composition has a SEC monomer percentage of greater than 94% (e.g., greater than 94.5%, 95%, 98%) when stored at 4°C for 6 months.

[0111] In some embodiments, the present disclosure provides a reconstituted solution comprising an anti-PD-1 antibody, which is obtained by reconstituting the aforementioned lyophilized formulation. The solution used for reconstitution includes, but is not limited to, water for injection, normal saline or dextrose solution, preferably water for injection.

[0112] In some embodiments, the present disclosure provides a reconstituted solution, which is a reconstituted form of the aforementioned lyophilized formulation.

[0113] In some embodiments, the present disclosure provides an article of manufacture comprising a container filled with the aforementioned pharmaceutical composition, lyophilized formulation or reconstituted solution. In some embodiments, the container is a neutral borosilicate glass tube manufactured injection vial.

[0114] The present disclosure also provides use of the foregoing pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture in the manufacture of a medicament for treating / preventing a disease or disorder.

[0115] The present disclosure also provides the foregoing pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture as a medicament for treating / preventing a disease or disorder.

[0116] The present disclosure also provides a method of treating or preventing a disease or disorder, comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of the foregoing pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture.

[0117] In some embodiments, the disease or disorder of any of the foregoing is a PD-1 -associated disease or disorder.

[0118] In some embodiments, the disease of any of the foregoing is a tumor. In other embodiments, the disease of any of the foregoing is selected from the group consisting of head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma; in some of these embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia; in other embodiments, the disease is selected from the group consisting of PD-L1 -positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, intestinal cancer, and colon cancer; in other embodiments, the disease is selected from the group consisting of melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, intestinal cancer, and colon cancer. BRIEF DESCRIPTION OF DRAWINGS

[0119] Figure 1 : Test results of anti-PD-1 antibodies blocking the binding of PD-1 to its ligands;

[0120] Figure 2 :The effect of anti-PD-1 antibody on IFNγ secretion by PBMC cells;

[0121] Figure 3 :Effect of anti-PD-1 antibody on MC38 colon cancer xenografts in mice;

[0122] Figure 4 :The effect of anti-PD-1 antibody on the tumor volume of mouse colon cancer MC38;

[0123] Figure 5 : DOE fitting diagram of anti-PD-1 antibody preparation. DETAILED DESCRIPTION

[0124] the term

[0125] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0126] "Buffer" or "buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH in an appropriate range include acetate buffers, succinate buffers, gluconate buffers, histidine buffers, oxalate buffers, lactate buffers, phosphate buffers, citrate buffers, tartrate buffers, fumarate buffers, glycylglycine buffers, and other organic acid buffers.

[0127] "Acetate buffer" or "acetate buffer" is a buffer comprising acetate ions. Examples of acetate buffers include acetate-sodium acetate, histidine-acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like. In some embodiments of the present disclosure, the acetate buffer is acetate-sodium acetate buffer (also known as sodium acetate, abbreviated as AA).

[0128] A "histidine salt buffer" or "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, and the like. In some embodiments of the present disclosure, the buffer is a histidine-acetate buffer (also known as histidine acetate, abbreviated His-AA).

[0129] A "succinate buffer" or "succinate buffer solution" is a buffer that includes succinate ions. Examples of succinate buffer solutions include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate buffer solutions, and the like. In some embodiments of the present disclosure, the succinate buffer is a succinate-sodium succinate buffer (also known as sodium succinate, abbreviated as SA).

[0130] A "citrate buffer" or "citrate buffer solution" is a buffer that includes citrate ions. Examples of citrate buffer solutions include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate buffer solutions, and the like. A preferred citrate buffer is a citrate-sodium citrate buffer (also known as sodium citrate, abbreviated as CA).

[0131] An "osmotic pressure adjusting agent" refers to a substance used to adjust the osmotic pressure of a solution. Examples of osmotic pressure adjusting agents include, but are not limited to, sugars (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like), amino acids (including arginine, glycine, cysteine, histidine, and the like), salts (sodium chloride, potassium chloride, calcium chloride, and the like). In some embodiments, the osmotic pressure adjusting agent is a sugar selected from the group consisting of: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol (also known as sorbose), mannitol, melibiose, rhamnose, melezitose, raffinose, stachyose, maltulose, lactulose, maltitol, sorbitol, maltitol, lactitol, and isomaltulose; in some embodiments, the sugar is a non-reducing disaccharide; in some embodiments, the sugar is preferably trehalose or sucrose, most preferably sucrose. In some embodiments, the osmotic pressure adjusting agent is an amino acid, preferably arginine, glycine; in some embodiments, the osmotic pressure adjusting agent is a salt, preferably sodium chloride.

[0132] "Isotonic" means that the formulation has essentially the same osmotic pressure as human blood. Isotonicity can be determined by methods known in the art, for example, by using a vapor pressure or freezing point osmometer. When the route of administration is subcutaneous injection, the osmotic pressure of the pharmaceutical composition is preferably controlled to be between 280 and 320 mOsm, and the osmotic pressure adjusting agent is preferably 70 to 90 mg / mL sucrose; more preferably, the osmotic pressure of the pharmaceutical formulation is controlled to be around 300 mOsm, and the osmotic pressure adjusting agent is preferably 80 mg / mL sucrose.

[0133] "Pharmaceutical composition" means a mixture of one or more of the compounds described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof with other chemical components, such as physiologically / pharmaceutically-acceptable carriers and excipients. The pharmaceutical composition retains the biological activity of the antibody active ingredient, facilitates administration to an organism, and promotes absorption of the active ingredient into the organism to thereby exhibit biological activity. In this context, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0134] "Substitution" means substitution of a solvent system for an antibody protein, for example, substitution of a high-salt or high-osmotic solvent system containing an antibody protein with a buffer system for a stable formulation by physical manipulation, thereby allowing the antibody protein to exist in a stable formulation. The physical manipulation includes, but is not limited to, ultrafiltration, dialysis, or resuspension after centrifugation.

[0135] The pharmaceutical composition or formulation of the present disclosure can be prepared by methods known in the art. For example, preparation of an antibody pharmaceutical composition or formulation: Step 1: Take a certain amount of purified antibody solution, and perform solvent substitution (preferably ultrafiltration) with a buffer containing no antibody (such as 10 mM sodium acetate buffer at pH 5.2), and substitute at least 6 times the volume of the ultrafiltration membrane, and concentrate the antibody to about 130 mg / mL. Add a certain volume of sucrose stock solution, mix well, and make the final sucrose concentration 80 mg / mL. Add a certain volume of polysorbate 80 stock solution, mix well, and make the final polysorbate 80 concentration 0.6 mg / mL. Add 10 mM sodium acetate buffer at pH 5.2 to constant volume, and make the antibody concentration 120 mg / mL (other formulations to be tested or stable formulations can be prepared according to similar procedures). After filtration, take a sample for sterility control. Pass the stock solution through a 0.22 μm filter, and collect the filtrate. Step 2: Adjust the fill volume, and fill the filtrate into a vial, and seal it, and take samples at the beginning, middle, and end of filling for fill volume difference control. Step 3: Start the capper, add an aluminum cap, and cap it. Step 4: Perform visual inspection, and confirm that the product has no fill volume inaccuracy and other defects. Print and paste the vial label; print the carton label, fold the carton, pack it, and paste the carton label.

[0136] The solution form of the pharmaceutical composition described in the present disclosure is water, unless otherwise specified.

[0137] "Lyophilized formulation" means a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step of a liquid or solution formulation. Lyophilized formulations can be obtained by freeze-drying a pharmaceutical composition or a liquid or solution formulation. Freeze-drying is carried out by freezing the formulation and subsequently subliming the water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic point or decomposition temperature of the formulation. The storage temperature of the product after primary drying is typically in the range of about -30 to 25°C (assuming the product remains frozen during primary drying), at a suitable pressure, typically in the range of about 50-250 mtorr. The size and type of the formulation, the container holding the sample (e.g., a glass vial), and the volume of the liquid determine the time required for drying, which can range from a few hours to several days (e.g., 40-60 hours). The secondary drying phase can be carried out at about 0-40°C, depending primarily on the type and size of the container and the type of protein employed. The secondary drying time is determined by the desired residual moisture level in the product, and typically requires at least about 5 hours. Typically, the water content of a lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure can be the same as that applied during the primary drying step, preferably, the pressure during secondary drying is lower than that of primary drying. The freeze-drying conditions can vary with the formulation and vial size.

[0138] The surfactant of the present disclosure can be selected from polysorbate 20, polysorbate 80, polyoxyl, Triton, sodium lauryl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, laurylamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyltaurate, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol, and the like. The preferred surfactant is polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0139] As used herein, the term "about" or "approximately" means that the value in question is within a range acceptable to one of ordinary skill in the art as determined by the particular value in question and how it is measured or determined (i.e., the limitations of the measuring system). For example, "about" can mean within one standard deviation or more than one standard deviation within 1. Alternatively, "about" or "approximately" can mean up to 20% of a range. For example, ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or less of the particular value after the term "about" or "approximately" based on the measuring method, technique conditions, detection reagents, and / or detection instruments commonly employed by one of skill in the art, such ranges are well known in the art. Furthermore, the term can mean up to one order of magnitude or up to 5 times a value, especially for biological systems or processes. Unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range of the particular value in question when that particular value appears in the application and claims.

[0140] The pharmaceutical compositions described herein can achieve a stable effect, wherein the antibody substantially retains its physical stability and / or chemical stability and / or biological activity after storage, preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. There are a variety of analytical techniques to measure protein stability, which can measure stability after storage for a selected period of time at a selected temperature. For example, a stable pharmaceutical antibody formulation is one in which no significant changes are observed: upon storage at refrigerated temperatures (2-8°C) for, e.g., at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. A stable formulation, e.g., by visual analysis, the pharmaceutical antibody formulation is colorless, or clear to slightly opalescent; the concentration, pH, and osmolality of the formulation have no more than ±10% change; generally no more than about 10%, preferably no more than about 5%, truncation is observed; generally no more than about 10%, preferably no more than about 5%, aggregation is formed, etc. In some embodiments, the pharmaceutical composition or lyophilized formulation of the present disclosure 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. In some embodiments, the relative stability of the formulation can be detected by observing the appearance of the formulation, SEC, non-reduced CE-SDS, and iCIEF, etc. under conditions of forced degradation (e.g., 40°C M1), accelerated conditions (e.g., 25°C M6), shaking D7 (e.g., 25°C, 300 rpm, shaking for 10 days), multiple freeze-thaw, etc.

[0141] An antibody "retains its physical stability" in a pharmaceutical formulation if it does not show a significant increase in aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines protein tertiary structure, and by FTIR spectroscopy, which determines protein secondary structure.

[0142] An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not show a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in conjunction with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0143] An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of manufacture of the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

[0144] The terms "Programmed Death 1," "Cellular Programmed Death 1," "Protein PD-1," "PD-1," "PDCD1," and "hPD-1" are used interchangeably and include variants, isoforms, species homologs of human PD-1, as well as analogs that share at least one common epitope with PD-1. The complete PD-1 sequence can be found under GenBank Accession No. U64863.

[0145] The term "Programmed Death Ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that, when bound to PD-1, downregulates T cell activation and cytokine secretion. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and 5 analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank Accession No. Q9NZQ7.

[0146] The term "cytokine" is a general term for proteins released by a cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include: human IL-2, IFN-gamma, IL-6, TNF alpha, IL-17, and IL-5.

[0147] The three letter code and one letter code for amino acids used herein are as described in J. Biol. Chem, 243, p 3558 (1968).

[0148] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, murine antibodies, chimeric antibodies, humanized antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity and specificity. The term "antibody" as used herein encompasses "full-length antibodies" and "antigen-binding fragments thereof. The anti-PD-1 antibody in some embodiments of the present disclosure is the anti-PD-1 antibody described in International Patent Application PCT / CN2020 / 074098, e.g., the "Hu23-11.IgG4AA" antibody. The entire contents of International Patent Application PCT / CN2020 / 074098 are incorporated herein by reference.

[0149] The terms "full-length antibody," "intact antibody," "complete antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, as distinct from an antigen-binding fragment as defined below.

[0150] The term "antigen binding fragment" or "functional fragment" is one or more fragments of an intact antibody that retain the ability to specifically bind to an antigen (e.g., PD-1). Examples of antigen binding fragments include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al. (1989) Nature 341 :544-546), which consists of a VH domain. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also included in the term "antigen binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same fashion as are intact antibodies. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulin. In some embodiments, antigen binding fragments of the disclosure include Fab, F(ab')2, Fab', single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), and the like.

[0151] The sequences of about 110 amino acids at the N-terminus of both the heavy and light chains are highly variable and constitute the variable region (V region); the remaining amino acids at the C-terminus are relatively invariant and constitute the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0152] The terms "complementarity determining region," "CDR," or "hypervariable region" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. Typically, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). The boundaries of the amino acid sequences of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al. (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003), and the like. For example, for the canonical format, following the Kabat convention, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia convention, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT convention, the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).The CDR regions of an antibody can be determined using the program IMGT / DomainGap Align, following IMGT rules. Following AbM rules, the CDR amino acid numbering in VH is 26-32 (HCDR1), 50-58 (HCDR2), and 95-102 (HCDR3); and in VL is 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Unless otherwise specified, the antibody variable region and CDR sequences referred to in the embodiments of the present disclosure apply to the "Kabat" numbering rules.

[0153] A "conventional variant" of the human antibody heavy chain constant region and human antibody light chain constant region described in the present disclosure refers to a variant of the heavy chain constant region or light chain constant region derived from human that does not change the structure and function of the antibody variable region, which has been disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that are subjected to site-directed modification and amino acid substitution, specific substitutions such as YTE mutation, L234A and / or L235A mutation, S228P mutation, and / or mutations to obtain a knob-into-hole structure (so that the antibody heavy chain has a combination of knob-Fc and hole-Fc), which have been proven to give the antibody new properties, but do not change the function of the antibody variable region.

[0154] The antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, preferably humanized antibodies.

[0155] The term "murine antibody" in the present disclosure refers to a monoclonal antibody against human PD-1 prepared according to the knowledge and skills in the art. When prepared, the test subject is injected with a PD-1 antigen, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. In a preferred embodiment of the present disclosure, the murine anti-PD-1 antibody can further comprise a light chain constant region of murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of murine IgG1, IgG2, IgG3 or variant thereof.

[0156] The term "chimeric antibody" is an antibody in which the variable region of a murine antibody is fused with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. Generally, to establish a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell, and the constant region gene of a human antibody is cloned as needed, and the murine variable region gene is linked to the human constant region gene to form a chimeric gene, which is then inserted into an expression vector, and finally the chimeric antibody is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the PD-1 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the PD-1 chimeric antibody further comprises a heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain constant region, or an IgG1, IgG2 or IgG4 variant using amino acid mutations (such as L234A and / or L235A mutations, and / or S228P mutations).

[0157] The term "humanized antibody" is also referred to as CDR-grafted antibody, which refers to an antibody in which the CDR sequences of a murine antibody are grafted into a human antibody variable region framework, i.e. an antibody generated by grafting different types of human germline antibody framework sequences. The heterogeneity reaction induced by the chimeric antibody due to carrying a large amount of murine protein components can be overcome. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. The germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase), and in Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. To avoid the decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal back or reverse mutations to maintain activity. The humanized antibody of the present disclosure also includes a humanized antibody further subjected to affinity maturation mutations of CDRs by yeast display.

[0158] The terms "human antibody," "human-derived antibody," "fully human antibody," "completely human antibody," as used herein interchangeably, have amino acid sequences corresponding to those produced by a human or human cell, or derived from a non-human source using a human antibody repertoire or other human antibody-encoding sequences. The definition expressly excludes humanized antibodies that contain non-human antigen binding residues. In some embodiments, fully human antibodies can be constructed by genetic or chromosomal transfection methods and phage display techniques, or from in vitro activated B cells, all of which are known in the art.

[0159] The term "amino acid difference" or "amino acid mutation" refers to the presence of an amino acid change or mutation in a variant protein or polypeptide as compared to a parent protein or polypeptide, including the insertion, deletion, or substitution of 1, 2, 3, or more amino acids from the parent protein or polypeptide.

[0160] The term "antibody framework" or "FR region" refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. In essence, it is the variable domain without the CDRs.

[0161] The term "epitope" or "antigenic determinant" refers to a site (e.g., a specific site on a PD-1 molecule) on an antigen that is specifically bound by an immunoglobulin or antibody. Epitopes are generally formed both by contiguous and non-contiguous amino acids. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

[0162] The terms "specifically binds," "selectively binds," "selectively binds to," and "binds specifically to" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody will bind to an antigen with an affinity (KD) of about less than 10 -7 M, for example, about less than 10 -9 M, 10 - 10 M, 10 -11 M or less.

[0163] The term "KD" or "Kd" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present disclosure will bind to an antigen with a KD of less than about 10 -7 M, for example, less than about 10 -8 M or 10 -9The dissociation equilibrium constant (KD) of M binding to PD-1, for example, as determined using surface plasmon resonance (SPR) technology in a BIACORE instrument.

[0164] When the term "compete" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it is meant that the antigen binding proteins compete with each other as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) the specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., a PD-1 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assay, solid phase direct label sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA with 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct label RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically the assay involves the use of purified antigen bound to a solid surface or cells that bears either the unlabeled test antigen binding protein and the labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Typically the test antigen binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding proteins that bind to an adjacent epitope in sufficient proximity to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder each other from binding. Additional details regarding methods for determining competitive binding are provided in the Examples herein. Typically when competing antigen binding proteins are present in excess, they will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more.In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0165] The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "operably linked" when they are functionally connected. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0166] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The vectors disclosed herein can be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be capable of integration into the genome of a host cell post-introduction into the host cell, and thereby be replicated along with the host genome (e.g., non-episomal mammalian vectors).

[0167] Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as in Current Protocols in Immunology, John Wiley & Sons, Inc., 5-8 and 15 chapters. For example, a mouse can be immunized with human PD-1 or a fragment thereof, the resulting antibodies can be renatured, purified, and can be subjected to amino acid sequencing using conventional methods. Antigen binding fragments can likewise be prepared using conventional methods. The antibodies or antigen binding fragments described herein are genetically engineered to have one or more human FR regions in the CDR regions of a non-human source. Human FR germline sequences can be obtained by alignment of the IMGT human antibody variable region germline gene database and MOE software, from the website of ImMunoGeneTics (IMGT) at http: / / imgt.cines.fr, or from the journal of immunology, 2001 ISBN 012441351.

[0168] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microbial hosts include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0169] The engineered antibodies or antigen binding fragments of the present disclosure can be produced and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expression of antibodies that specifically bind to human PD-1. Positive clones are expanded in serum-free media in a bioreactor to produce the antibodies. The culture fluid in which the antibodies are secreted can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column with a modified buffer. Non-specifically bound components are washed away. The bound antibodies are eluted using a pH gradient method, and the antibody fragments are detected using SDS-PAGE and collected. The antibodies can be filter concentrated using conventional methods. Soluble aggregates and multimers can also be removed using conventional methods, such as molecular sieving, ion exchange. The resulting product is immediately frozen, such as at -70°C, or lyophilized.

[0170] "Administering," "administered," and "treatment" when applied to an animal, human, test subject, cell, tissue, organ, or biological fluid, means the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, test subject, cell, tissue, organ, or biological fluid. "Administering," "administered," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of the reagent with the cell, as well as contact of the reagent with a fluid that is in contact with the cell. "Administering," "administered," and "treatment" also mean treatment by a reagent, diagnostic, binding composition, or by another cell in vitro and ex vivo, for example. "Treatment" when applied to a human, veterinary, or research subject, means therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0171] "Treatment" means the administration of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the disclosure, to a patient having one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as "therapeutically effective amount") can vary depending on factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical test method typically used by a physician or other professional health care provider to assess the severity or progression of the symptom. While embodiments of the disclosure (e.g., a method of treatment or article of manufacture) can not be effective in alleviating each target disease symptom, it is determined that the target disease symptom should be alleviated in a statistically significant number of patients according to any statistical test method known in the art, such as the Student t-test, the Chi-square test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0172] A "conservative modification" or "conservative substitution or replacement" is the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. As is known to those skilled in the art, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). In addition, substitutions of amino acids with similar properties are not likely to disrupt biological activity. Exemplary conservative substitutions are set forth in the table "Exemplary Amino Acid Conservative Substitutions" below.

[0173] Table 3. Exemplary Amino Acid Conservative Substitutions

[0174] Original Residue Conservative Substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln; His; Asp

[0175] Asp (D) Glu; Asn Cys (C) Ser; Ala; Val Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

[0176] An "effective amount" or "effective dose" is the amount of a drug, compound, or pharmaceutical composition necessary to achieve a desired therapeutic result. For prophylactic use, a desired result includes eliminating or reducing a risk, lessening the severity, or delaying the onset of a disorder, including biochemical, histological, and / or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes presenting during the course of the disorder. For therapeutic applications, a desired result includes a clinical result, such as reducing the incidence of, or ameliorating one or more symptoms of, a disorder associated with a target antigen of the disclosure, reducing the dosage of other medications required to treat a disorder, enhancing the effect of another medication, and / or delaying the progression of a disorder associated with a target antigen of the disclosure in a patient.

[0177] "Exogenous" refers to a substance produced outside of a cell, organism, or human body, as the case can be. "Endogenous" refers to a substance produced inside of a cell, organism, or human body, as the case can be.

[0178] "Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions in the comparison times 100. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. If 95 of 100 positions in two sequences are matched or homologous, then the two sequences are 95% homologous. Typically, the comparison is performed using the best alignment of two sequences, e.g., the two sequences are optimally aligned to give the maximum percent homology. The comparison can be performed by the BLAST algorithm, for example, where the algorithm parameters are selected to give the maximum match between the two sequences over the entire length of the respective reference sequences. The following references are directed to the BLAST algorithm, which is frequently used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410; Gish, W. et al. (1993) Nature Genet. 3:266-272; Madden, T.L. et al. (1996) Meth. Enzymol. 266:131-141; Altschul, S.F. et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other routine BLAST algorithms, such as those provided by NCBI BLAST, are also well known to those of skill in the art.

[0179] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived from it, without regard to the number of transfers. It is also understood that all progeny can not necessarily be identical to the parental cell since there can be, as a result of mutation, some genetic alteration in the progeny. The alteration is generally within acceptable limits to give a desired characteristic to the cell to be used in the production of the desired product.

[0180] As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a minute quantity of a specific portion of nucleic acid, RNA and / or DNA, is amplified as described in, for example, U.S. Patent No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond is required so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotides of the two primers can be identical to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, and the like. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol. 51 :263; Erlich, ed. (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used herein is considered an example of, but not the only example of, a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which method includes the use of known nucleic acids as primers and a nucleic acid polymerase to amplify or generate a specific portion of a nucleic acid.

[0181] "Isolated" refers to the state of purification and in this context means that the specified molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cellular debris and growth media. Generally, the term "isolated" is not intended to mean completely free from these materials or free from water, buffers, or salts, unless they are present in amounts that significantly interfere with experimental or therapeutic uses of the compounds as described herein.

[0182] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes situations where the event or circumstance occurs and situations where it does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of the specified sequence can or can not be present.

[0183] Further, the present disclosure includes medicaments for treating a disease associated with cells positive for a target antigen (e.g., PD-1), the medicaments comprising an anti-PD-1 antibody of the present disclosure as an active ingredient.

[0184] The disease associated with PD-1 in the present disclosure is not limited as long as it is a disease associated with PD-1, for example, a therapeutic response induced by the molecules of the present disclosure can be through binding to human PD-1, then blocking the binding of PD-1 to its ligand PD-L1, PD-L2, or killing tumor cells overexpressing PD-1. Therefore, the molecules of the present disclosure are very useful for those who have tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, non-small cell lung cancer, bladder cancer, etc. when in preparations and formulations suitable for therapeutic applications.

[0185] In addition, the present disclosure relates to a method for immunoassay or measurement of a target antigen (e.g., PD-1), a reagent for immunoassay or measurement of a target antigen (e.g., PD-1), a method for immunoassay or measurement of a cell expressing a target antigen (e.g., PD-1), and a diagnostic agent for diagnosing a disease associated with a target antigen (e.g., PD-1) positive cell, which comprises an antibody or antibody fragment of the present disclosure that specifically recognizes a target antigen (e.g., human PD-1) and binds to the amino acid sequence or three-dimensional structure of the extracellular region as an active ingredient.

[0186] In the present disclosure, the method for detecting or measuring the amount of a target antigen (e.g., PD-1) can be any known method. For example, it includes an immunoassay or measurement method.

[0187] The immunoassay or measurement method is a method for detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunoassay or measurement method include a radioisotope-labeled immunological antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a fluorescent immunoassay (FIA), a luminescent immunoassay, a Western blotting method, a physical and chemical method, etc.

[0188] The above-mentioned disease associated with PD-1 positive cells can be diagnosed by detecting or measuring cells expressing PD-1 with the antibody or antibody fragment of the present disclosure.

[0189] To detect cells expressing a polypeptide, known immunoassay methods can be used, and preferably, immunoprecipitation, fluorescent cell staining, immunohistological staining, etc. can be used. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) or the like can be used.

[0190] In the present disclosure, the living sample for detecting or measuring a target antigen (e.g., PD-1) is not particularly limited as long as it has the possibility of containing cells expressing a target antigen (e.g., PD-1), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

[0191] The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure can further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction according to the required diagnostic method. The reagent for performing an antigen-antibody reaction includes a buffer, a salt, etc. The reagent for detection includes a reagent generally used in an immunoassay or assay method, such as a labeled second antibody recognizing the monoclonal antibody, the antibody fragment thereof, or a conjugate thereof, and a substrate corresponding to the label, etc.

[0192] In some embodiments, the antigen is prepared as follows:

[0193] A human PD-1-IgG1 Fc fusion protein was designed and synthesized, in which the N-terminus is the extracellular region of 150 amino acids of human PD-1 and the C-terminus is the Fc segment of human IgG1 (hIgG1 Fc). The recombinant PD-1-Fc protein with high purity was obtained by purification with a Protein A affinity column and was used to detect the binding of anti-PD-1 antibody to the antigen.

[0194] Human PD-1-IgG1 Fc (SEQ ID NO: 1):

[0195]

[0196] Note: The underlined part is the signal peptide, the normal part is the extracellular region of human PD-1, and the italic part is hIgG1 Fc Signal Peptide Hu32 VH1 + extracellular region + hIgG1 Fc.

[0197] Human PD-1-his (SEQ ID NO: 2):

[0198]

[0199] Transfected cell nucleic acid encoded PD-1 antigen (SEQ ID NO: 3):

[0200]

[0201] In some embodiments, the anti-human PD-1 antibody can be produced by immunizing mice, and can also be obtained from an anti-human PD-1 phage mouse immunization library.

[0202] The method for preparing an anti-human PD-1 antibody by immunizing mice is as follows:

[0203] 1. Immunization: SJL white mice, female, 6-8 weeks old and Balb / c white mice, female, 6-8 weeks old were used for the experiment. The feeding environment: SPF level. After the mice were purchased, they were fed in the laboratory environment for 1 week, adjusted to 12 / 12 hours light / dark cycle, temperature 20-25°C; humidity 40-60%. The mice that had adapted to the environment were immunized according to different schemes, 6-10 mice in each group. The immunization antigen can be purified recombinant protein PD-1-IgG1Fc (see SEQ ID NO: 1), PD-1-his (see SEQ ID NO: 2), or PD-1 as an antigen (see SEQ ID NO: 3) transfected Jurkat / CHO-PD-1 cells, and a single antigen can be used with different immunization adjuvants or different types of immunogens for cross immunization. The immunization site can be the abdominal cavity or the back subcutaneously, or alternately immunized at both sites. Gold Adjuvant (hereinafter referred to as Titermax, purchased from Sigma, product number T2684) and Imject Alum Adjuvant (hereinafter referred to as Alum, purchased from Pierce, product number 77161) were cross-immunized. The ratio of antigen to adjuvant (Titermax) was 1:1, the ratio of antigen to adjuvant (Alum) was 3:1, 25-50 μg / mouse (primary immunization), 50 μg / mouse (boosting), or 1 x 107 Jurkat / CHO-PD-1 cells / mouse. Intraperitoneal injection of 25-50 μg / mouse of emulsified antigen on day 0, once a week or once every two weeks after the primary immunization, Titermax and Alum were used alternately, a total of 5-8 times.

[0204] 2. Cell fusion: Mice with high antibody titers in serum were selected for spleen cell fusion. The mice were bled from the eye 72 hours after the booster immunization ("h" is the abbreviation of "hour", the same below), and were killed by cervical dislocation and placed in 75% ethanol for disinfection. The spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (Chinese Academy of Sciences) by optimized PEG-mediated fusion steps to obtain hybridoma cells. The fused hybridoma cells were resuspended with HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 x HAT and 1 x OPI), and were dispensed into 96-well cell culture plates (1 x 105 / 150 μL / well), incubated at 37°C, 5% CO2, and seeded in 10-30 plates. On the 5th day after fusion, HAT complete medium was added, 50 μL / well, 37°C, 5% CO2 incubation. From the 7th day to the 8th day after fusion, according to the cell growth density, the medium was changed to 200 μL / well, 37°C, 5% CO2 incubation.

[0205] 3. Hybridoma cell screening: 7-9 days after fusion, according to the cell growth density, the positive cell wells were detected by ELISA method for antibody binding to PD-1, and the positive cell wells were detected by blocking ELISA for PD-1 / PDL1 binding. The positive cell wells were replaced with new liquid, and the cell density was expanded to a 24-well plate in time. The cell strains transferred to the 24-well plate were preserved and subcloned for the first time after retesting. The positive cells screened by the first subcloning were preserved and subcloned for the second or third time until single cell clones were obtained. Multiple fusions obtained hybridoma cells with blocking effect of PD-1 and PDL1 binding.

[0206] The method for obtaining anti-human PD-1 antibody by anti-human PD-1 phage mouse immunization library is as follows:

[0207] 1. Construction of anti-human PD-1 phage mouse immunization library: The spleen of a mouse with high antibody titer in serum was selected, and total RNA of the tissue was extracted by Trizol (Invitrogen Cat No. 15596-018). Reverse transcription was performed by using PrimeScript TM II 1stStrand cDNA Synthesis Kit kit (Takara Cat No. 6210A). The primers for constructing the library were designed and synthesized according to the IMGT database. Single-chain antibody fragments were obtained by three rounds of PCR reaction. The single-chain antibody fragments and the modified library construction vector pCantab5E (Amersham Biosciences / GE Cat No. 27-9400-01) were subjected to enzyme digestion with Sfi1 (NEB Cat No. #R0123L), and then purified and recovered by using Gel Extraction Kit (Omega Cat No. D2500-02) after electrophoresis. Then, the T4 DNA ligase (NEB Cat No. #M0202L) was used for ligation at 16°C for 16-18 hours, and then purified and recovered by using the above kit, and finally eluted with deionized water. 1 μg of the ligation product was mixed with 1 of the electrocompetent TG1 (Lucigen Cat No. 60502-2), and the parameters of the electrotransformation instrument (Bio Rad Micropulser) were set to 2.5 kV, 200 Ω, and 25 uF for electrotransformation. The transformation was repeated for 10 times, and then plated and cultured at 37°C for 16-18 hours. All the colonies were scraped and mixed together, and then 15% glycerol was added, and then stored at -80°C for standby.

[0208] 2. Screening of anti-human PD-1 phage mouse immunization library: The packaged anti-human PD-1 phage mouse immunization library (1×10 12 -1×10 13) with 100 μL streptavidin microbeads (Miltenyi Biotec, Auburn, CA) in 1 mL of 2% skim milk-phosphate buffered saline (abbreviated as MPBS) for 1 hour at room temperature, placed on a magnetic stand, and the supernatant was removed. The supernatant was added to 10 μg / mL biotinylated human PD-1-ECD-his protein (purchased from Sino Biological) for 1 hour at room temperature, and then 100 μL of streptavidin-coated magnetic beads (1 mL of MPBS pre-incubation) were added for 1 hour at room temperature. It was loaded on a magnetic stand system for sorting, and the supernatant was removed. 1 mL of PBST (phosphate buffered saline containing 0.1% Tween-20) was added, and the tube was inverted several times, and after the supernatant was removed, fresh washing solution was added, and the process was repeated 11 times to remove unbound antibody fragments. 0.5 mL of elution solution (50 μL of 10 mg / mL trypsin stock solution (stock solution) was added to 450 μL of PBS). Shake at room temperature for 15 min. Place on a magnetic stand, and the supernatant was removed to a new EP tube. TG1 was inoculated into 2YT medium and amplified to a bacterial density of OD600=0.4. 1.75 mL of TG1 (OD600=0.4) was added to each tube, and 250 μL of eluted phage was added. Incubate at 37°C in a water bath for 30 min. Gradient dilution and plating were used for titer testing. The rest of the TG1 solution was centrifuged and plated, and incubated at 37°C overnight.

[0209] The phage mouse immune library was screened by 2-3 rounds of MACS (streptomycin magnetic beads, Invitrogen) using biotinylated human PD-1-ECD-his antigen, and finally obtained monoclonal antibodies with binding to PD-1 and blocking the binding of PD-1 to PD-L1. Sequencing verification was performed to obtain the variable region sequence of the antibody.

[0210] In some embodiments, the purification method of the antibody or antigen protein is as follows:

[0211] 1. Hybridoma supernatant separation and purification / Protein G affinity chromatography:

[0212] For purification of mouse hybridoma supernatant, Protein G affinity chromatography is preferred. The supernatant is obtained by centrifugation of the culture. The pH of the supernatant is adjusted by adding 10-15% volume of 1M Tris-HCl (pH 8.0-8.5). The Protein G column is washed with 6M guanidine hydrochloride for 3-5 column volumes, and then with pure water for 3-5 column volumes. The column is equilibrated with 1x PBS (pH 7.4) for 3-5 column volumes. The supernatant is loaded onto the column at a low flow rate, and the flow rate is controlled so that the retention time is about 1 min or longer. The column is washed with 1x PBS (pH 7.4) for 3-5 column volumes until the UV absorption returns to the baseline. The sample is eluted with 0.1M acetic acid / sodium acetate (pH 3.0) buffer, and the elution peak is collected according to the UV detection. The eluted product is temporarily stored by adjusting the pH to 5-6 with 1M Tris-HCl (pH 8.0). The eluted product can be replaced into the desired buffer system by methods known to those skilled in the art, such as ultrafiltration with a tube, or desalting with G-25, or removing the aggregate components from the eluted product by using a high-resolution molecular exclusion column such as Superdex 200 to improve the purity of the sample.

[0213] 2. Protein A affinity chromatography for purification of proteins or antibodies:

[0214] The supernatant of the cell culture expressing the antigen protein or antibody is first centrifuged to obtain the supernatant. The Protein A affinity column is washed with 6M guanidine hydrochloride for 3-5 column volumes, and then with pure water for 3-5 column volumes. The column is equilibrated with 1x PBS (pH 7.4) for 3-5 column volumes. The supernatant is loaded onto the column at a low flow rate, and the flow rate is controlled so that the retention time is about 1 min or longer. After the binding is completed, the column is washed with 1x PBS (pH 7.4) for 3-5 column volumes until the UV absorption returns to the baseline. The sample is eluted with 0.1M acetic acid / sodium acetate (pH 3.0-3.5) buffer, and the elution peak is collected according to the UV detection. The eluted product is temporarily stored by adjusting the pH to 5-6 with 1M Tris-HCl (pH 8.0). The eluted product can be replaced into the desired buffer system by methods known to those skilled in the art, such as ultrafiltration with a tube, or desalting with G-25, or removing the aggregate components from the eluted product by using a high-resolution molecular exclusion column such as Superdex 200 to improve the purity of the sample.

[0215] The present disclosure will be further described with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure. The experimental methods in the present disclosure examples, unless otherwise specified, are generally performed according to conventional conditions, such as Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; or Molecular Cloning: A Laboratory Manual; or according to the conditions recommended by the manufacturer of the materials or products. Reagents, unless otherwise specified, are commercially available and are conventional reagents.

[0216] Example 1. Anti-human PD-1 murine antibody acquisition

[0217] The anti-human PD-1 murine antibodies obtained by the foregoing method were subjected to antigen binding experiments, and a plurality of antibodies with good activity were screened, including M23, M32 and M33. Single cell clones were expanded and cultured, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using mouse-Ig degenerate primers to obtain the variable region sequence of the antibody. The murine antibody variable region sequence was linked to the human antibody constant region sequence, and the chimeric antibody of the murine monoclonal antibody was cloned and recombinantly expressed, and in vitro activity experiments were performed to confirm that the obtained monoclonal antibody variable region sequence was correct.

[0218] The variable region sequences of the murine antibodies M23, M32 and M33 were measured as follows:

[0219] Heavy chain variable region of murine antibody M23 (SEQ ID NO: 4):

[0220]

[0221] Light chain variable region of murine antibody M23 (SEQ ID NO: 5):

[0222]

[0223] Heavy chain variable region of murine antibody M32 (SEQ ID NO: 6):

[0224]

[0225] Light chain variable region of murine antibody M32 (SEQ ID NO: 7):

[0226]

[0227] Heavy chain variable region of murine antibody M33: (SEQ ID NO: 19)

[0228]

[0229] Light chain variable region of murine antibody M33: (SEQ ID NO: 20)

[0230]

[0231] Note: In the heavy chain variable region and light chain variable region sequences of the above antibodies, the underlined sequences are the CDR sequences determined by the Kabat numbering system, in turn FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0232] Table 4. Murine antibody M23, M32 and M33 heavy chain and light chain CDR region sequences

[0233]

[0234]

[0235] Note: The CDR sequences of the antibodies in the table are determined according to the Kabat numbering system.

[0236] Example 2. Humanization of anti-human PD-1 monoclonal antibodies

[0237] By comparing the IMGT human antibody heavy and light chain variable region gene database and MOE software analysis, respectively, the human germline heavy and light chain variable region genes with high sequence identity to the light and heavy chain sequences of M23, M32 and M33 were selected as templates, and the CDRs of the three murine antibodies were transplanted into the corresponding human antibody templates, respectively, to construct the corresponding humanized antibodies.

[0238] 1. Humanization of murine antibody M23

[0239] 1.1 Selection of humanization framework of murine antibody M23

[0240] The humanized light chain template of murine antibody M23 is IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain template is IGHV1-69*02 and IGHJ6*01. The variable region sequence after humanization is as follows (the underlined sequences are CDR sequences):

[0241] Hu23 VH-CDR grafting: (SEQ ID NO: 27)

[0242]

[0243] Hu23 VL-CDR grafting: (SEQ ID NO: 28)

[0244]

[0245] 1.2 Selection of humanization template and reverse mutation design of murine antibody M23

[0246] Table 5. Reverse mutations of humanized antibodies of murine antibody M23

[0247]

[0248]

[0249] Note: Grafted represents the implantation of mouse antibody CDRs into human germline FR sequences. Amino acid residues are identified and annotated using the Kabat numbering system. For example, I2G indicates that the second position (I) in the Kabat numbering system has been mutated back to G.

[0250] The sequences of the light and heavy chain variable regions of the humanized antibody M23 are as follows:

[0251] >Hu23VL1 (same as Hu23VL-CDR grafted): (SEQ ID NO: 28)

[0252]

[0253] >Hu23VL2 (SEQ ID NO: 29)

[0254]

[0255] >Hu23VH1 (same as Hu23VH-CDR grafted): (SEQ ID NO: 27)

[0256]

[0257] >Hu23VH2 (SEQ ID NO: 30)

[0258]

[0259] >Hu23VH3 (SEQ ID NO: 31)

[0260]

[0261] >Hu23VH4 (SEQ ID NO: 32)

[0262]

[0263] 1.3 Humanized sequence combination of mouse antibody M23

[0264] The antibodies obtained after humanization of the mouse antibody M23 and their variable region combinations are shown in the table below.

[0265] Table 6. Combinations of humanized Hu23 antibody variable regions

[0266]

[0267] Note: "Hu23-1" refers to an antibody having a light chain variable region of Hu23 VL1 and a heavy chain variable region of Hu23 VH1, and so on for the other combinations.

[0268] The antibody light / heavy chain variable region combinations referred to in the above table (e.g., Hu23-1) can be linked to antibody light / heavy chain constant regions to form full-length antibodies; in the disclosure, unless otherwise specified, when the light chain variable region is linked to the Kappa chain constant region set forth in SEQ ID NO:73 to form an antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region set forth in SEQ ID NO:72 or the IgG4-P heavy chain constant region set forth in SEQ ID NO:79 to form an antibody heavy chain, the full-length antibody formed is indicated by the name of the antibody light / heavy chain variable region combination referred to in the table (e.g., Hu23-1) with the suffix ".IgG4AA" if the full-length antibody is formed with the IgG4-AA heavy chain constant region, or with the suffix ".IgG4P" if the full-length antibody is formed with the IgG4-P heavy chain constant region, e.g., "Hu23-1.IgG4AA" indicates a full-length antibody formed with a heavy chain comprising the Hu23 VH1 heavy chain variable region linked to the IgG4-AA heavy chain constant region set forth in SEQ ID NO:72, and a light chain comprising the Hu23 VL1 light chain variable region linked to the Kappa chain constant region set forth in SEQ ID NO:73. "Hu23-1.IgG4P" indicates a full-length antibody formed with a heavy chain comprising the Hu23 VH1 heavy chain variable region linked to the IgG4-P heavy chain constant region set forth in SEQ ID NO:79, and a light chain comprising the Hu23 VL1 light chain variable region linked to the Kappa chain constant region set forth in SEQ ID NO:73.

[0269] 2. Humanization of murine antibody M32

[0270] 2.1 Selection of framework for humanization of murine antibody M32

[0271] The humanized light chain template for murine antibody M32 is IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain template is IGHV1-69*02 and IGHJ6*01, and the humanized variable region sequences are as follows (underlined are CDR sequences):

[0272] Hu32 VH-CDR graft: (SEQ ID NO:33) IGHV1-69*02 and IGHJ6*01

[0273]

[0274] Hu32 VL-CDR graft: (SEQ ID NO:34)

[0275]

[0276] 2.2 Selection of humanization templates for murine antibody M32 and design of back-mutations

[0277] Table 7. Back-mutations of humanized antibodies of murine antibody M32

[0278]

[0279]

[0280] Note: Grafted represents murine antibody CDRs grafted into human germline FR region sequences. Amino acid residues are identified and annotated by the Kabat numbering system, e.g., I2V means mutating I at Kabat position 2 to V according to the Kabat numbering system.

[0281] The humanized antibody light and heavy chain variable region sequences of murine antibody M32 are as follows:

[0282] Hu32 VL1 (same as Hu32 VL-CDR grafted): (SEQ ID NO: 34)

[0283]

[0284] Hu32 VL2 (SEQ ID NO: 35)

[0285]

[0286] Hu32 VH1 (same as Hu32 VH-CDR grafted): (SEQ ID NO: 33)

[0287]

[0288] Hu32 VH2 (SEQ ID NO: 36)

[0289]

[0290] Hu32 VH3 (SEQ ID NO: 37)

[0291]

[0292] Hu32 VH4 (SEQ ID NO: 38)

[0293]

[0294] Hu32 VH5 (SEQ ID NO: 39)

[0295]

[0296] Hu32 VH6 (SEQ ID NO: 40)

[0297]

[0298] 2.3 Humanized sequence combinations of murine antibody M32

[0299] Antibodies obtained after humanization of murine antibody M32 and variable region combinations thereof.

[0300] Table 8. Humanized antibody Hu32 light / heavy chain variable region combinations

[0301]

[0302] Note: In the table, for example, "Hu32-1" refers to an antibody light / heavy chain variable region combination in which the light chain variable region is Hu32 VL1 and the heavy chain variable region is Hu32 VH1, and so on.

[0303] The antibody light / heavy chain variable region combinations referred to in the above table (e.g. Hu32-1) can be linked to antibody light / heavy chain constant regions, respectively, to form full-length antibodies; in the present disclosure, unless explicitly stated otherwise, when forming full-length antibodies, the light chain variable region is linked to the Kappa chain constant region shown in SEQ ID NO: 73 to form an antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or the IgG4-P heavy chain constant region shown in SEQ ID NO: 79 to form an antibody heavy chain, and the name of the antibody light / heavy chain variable region combination referred to in the table (e.g. Hu32-1) is suffixed with ".IgG4AA" to indicate a full-length antibody formed with the IgG4-AA heavy chain constant region, or suffixed with ".IgG4P" to indicate a full-length antibody formed with the IgG4-P heavy chain constant region, for example, "Hu32-1.IgG4AA" indicates a full-length antibody formed with a heavy chain comprising a heavy chain variable region and an IgG4-AA heavy chain constant region as shown in SEQ ID NO: 72, and a light chain comprising a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. Hu32 VL1 a heavy chain comprising a heavy chain variable region and an IgG4-AA heavy chain constant region as shown in SEQ ID NO: 72, and a light chain comprising a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. Hu32 VH1 a heavy chain comprising a heavy chain variable region and an IgG4-P heavy chain constant region as shown in SEQ ID NO: 79, and a light chain comprising a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. Hu32 VL1 a heavy chain comprising a heavy chain variable region and an IgG4-P heavy chain constant region as shown in SEQ ID NO: 79, and a light chain comprising a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. Hu33 VH3 a heavy chain comprising a heavy chain variable region and an IgG4-P heavy chain constant region as shown in SEQ ID NO: 79, and a light chain comprising a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73.

[0304] 3. Humanization of murine antibody M33

[0305] 3.1 Selection of frameworks for humanization of murine antibody M33

[0306] The humanized light chain template for murine antibody M33 is IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain template is IGHV3-7 and IGHJ6*01. The humanized variable region sequences are as follows:

[0307] Hu33 VH-CDR Grafted (SEQ ID NO: 41):

[0308]

[0309] Hu33 VL-CDR Grafted (SEQ ID NO: 42):

[0310]

[0311] 3.2 Selection of humanized templates for murine antibody M33 and design of backmutations

[0312] Table 9. Backmutations of humanized antibodies of murine antibody M33

[0313]

[0314] Note: Grafted represents murine antibody CDRs grafted into human germline FR region sequences. Amino acid residues are determined and annotated by the Kabat numbering system, e.g. F71Y means mutating F at position 71 according to Kabat numbering to Y.

[0315] The humanized light chain variable region and heavy chain variable region sequences of murine antibody M33 are as follows:

[0316] Hu33 VL1 (same as Hu33 VL-CDR grafted): (SEQ ID NO: 42)

[0317]

[0318] Hu33 VL2 (SEQ ID NO: 43)

[0319]

[0320] Hu33 VL3 (SEQ ID NO: 44)

[0321]

[0322] Hu33 VH1 (same as Hu33 VH-CDR grafted): (SEQ ID NO: 41)

[0323]

[0324] Hu33VH2 (SEQ ID NO: 45)

[0325]

[0326] Hu33VH3 (SEQ ID NO: 46)

[0327]

[0328]

[0329] 3.3 Humanized sequence combinations of murine antibody M33

[0330] Table 10. Humanized antibody light / heavy chain variable region combinations

[0331]

[0332] Note: For example, "Hu33-6" in the table refers to an antibody light / heavy chain variable region combination in which the light chain variable region is Hu33 VL2 and the heavy chain variable region is Hu33 VH3, and so on.

[0333] The antibody light / heavy chain variable region combinations referred to in the above table (e.g., Hu33-6) can be linked to antibody light / heavy chain constant regions, respectively, to form full-length antibodies; in the present disclosure, unless otherwise specified, when a full-length antibody is formed, the light chain variable region is linked to the Kappa chain constant region shown in SEQ ID NO: 73 to form an antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or the IgG4-P heavy chain constant region shown in SEQ ID NO: 79 to form an antibody heavy chain, and the name of the antibody light / heavy chain variable region combination referred to in the table (e.g., Hu33-6) is suffixed with ".IgG4AA" to indicate a full-length antibody formed with the IgG4-AA heavy chain constant region, or suffixed with ".IgG4P" to indicate a full-length antibody formed with the IgG4-P heavy chain constant region, for example, "Hu33-6.IgG4AA" indicates a full-length antibody formed with a heavy chain variable region and an IgG4-AA heavy chain constant region as shown in SEQ ID NO: 72, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. Hu33 VL2 a heavy chain formed with a heavy chain variable region and an IgG4-AA heavy chain constant region as shown in SEQ ID NO: 72, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. "Hu33-6.IgG4P" indicates a full-length antibody formed with a heavy chain variable region and an IgG4-P heavy chain constant region as shown in SEQ ID NO: 79, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. Hu33 VH3 a heavy chain formed with a heavy chain variable region and an IgG4-AA heavy chain constant region as shown in SEQ ID NO: 72, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. "Hu33-6.IgG4P" indicates a full-length antibody formed with a heavy chain variable region and an IgG4-P heavy chain constant region as shown in SEQ ID NO: 79, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. Hu33 VL2 a heavy chain formed with a heavy chain variable region and an IgG4-AA heavy chain constant region as shown in SEQ ID NO: 72, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. "Hu33-6.IgG4P" indicates a full-length antibody formed with a heavy chain variable region and an IgG4-P heavy chain constant region as shown in SEQ ID NO: 79, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. Figure 1 a heavy chain formed with a heavy chain variable region and an IgG4-AA heavy chain constant region as shown in SEQ ID NO: 72, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73. "Hu33-6.IgG4P" indicates a full-length antibody formed with a heavy chain variable region and an IgG4-P heavy chain constant region as shown in SEQ ID NO: 79, and a light chain formed with a light chain variable region and a Kappa chain constant region as shown in SEQ ID NO: 73.

[0334] 4. Mutants of humanized antibodies

[0335] 4.1 Mutant antibodies of Hu23 humanized antibodies

[0336] By computer simulation, site-directed mutagenesis was performed on the specific site amino acids of the light chain LCDR1 (SEQ ID NO: 11) of the Hu23 humanized antibody, and the specific mutations are shown in Table 11:

[0337] Table 11. Mutant sequences of Hu23 light chain LCDR1:

[0338]

[0339]

[0340] Note: Hu23 LCDR1 (N28Q) represents the mutant sequence of LCDR1 in which the 28th N of Hu23 humanized antibody light chain variable region Hu23 VL1 or Hu23 VL2 is mutated to Q according to Kabat numbering rules, and Hu23 LCDR1 (G29A) represents the mutant sequence of LCDR1 in which the 29th G of Hu23 humanized antibody light chain variable region Hu23 VL1 or Hu23 VL2 is mutated to A according to Kabat numbering rules (CDRs are determined by Kabat numbering system).

[0341] The sequence of the Hu23 humanized antibody light chain variable region after mutation of LCDR1 is as follows:

[0342] The sequence of Hu23 VL1 (N28Q) is:

[0343]

[0344] The sequence of Hu23 VL1 (N28L) is:

[0345]

[0346] The sequence of Hu23 VL1 (N28T) is:

[0347]

[0348] The sequence of Hu23 VL1 (N28D) is:

[0349]

[0350] The sequence of Hu23 VL1 (G29A) is:

[0351]

[0352] The Hu23 VL1 (G29V) sequence is:

[0353]

[0354] The Hu23 VL2 (N28Q) sequence is:

[0355]

[0356] The Hu23 VL2 (N28L) sequence is:

[0357]

[0358] The Hu23 VL2 (N28T) sequence is:

[0359]

[0360] The Hu23 VL2 (N28D) sequence is:

[0361]

[0362] The Hu23 VL2 (G29A) sequence is:

[0363]

[0364] The Hu23 VL2 (G29V) sequence is:

[0365]

[0366] Table 12. Hu23 humanized antibody light / heavy chain variable region combinations

[0367]

[0368] Note: For example, "Hu23-11" in the table refers to an antibody light / heavy chain variable region combination in which the light chain variable region is Hu23 VL1 (N28T) and the heavy chain variable region is Hu23 VH1, and so on.

[0369] The antibody light / heavy chain variable region combinations referred to in the above table (e.g. Hu23-11) can be linked to antibody light / heavy chain constant regions to form full length antibodies; in the present disclosure, unless otherwise specified, when forming full length antibodies, the light chain variable region is linked to the Kappa chain constant region shown in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or the IgG4-P heavy chain constant region shown in SEQ ID NO: 79 to form the antibody heavy chain, and the name of the antibody light / heavy chain variable region combination referred to in the table (e.g. Hu23-11) is suffixed with “.IgG4AA” to indicate the full length antibody formed with the IgG4-AA heavy chain constant region, or suffixed with “.IgG4P” to indicate the full length antibody formed with the IgG4-P heavy chain constant region, for example, “Hu23-11.IgG4AA” indicates a full length antibody formed by the Hu23VH1 heavy chain variable region and the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, and the light chain formed by the Hu23VL1(N28T) light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73. “Hu23-11.IgG4P” indicates a full length antibody formed by the Hu23VH1 heavy chain variable region and the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and the light chain formed by the Hu23VL1(N28T) light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73.

[0370] The experimental results show that the humanized antibodies of Hu23LCDR1(N28Q), Hu23LCDR1(N28L), Hu23LCDR1(N28T), Hu23LCDR1(N28D), Hu23LCDR1(G29A), and Hu23LCDR1(G29V) all maintain the binding ability to PD-1 (Table 16).

[0371] 4.2 Mutated antibodies of Hu32 humanized antibodies

[0372] Through sequence analysis, the sequences of the series of humanized antibodies Hu23 derived from M23 and the series of humanized antibodies Hu32 derived from M32 have high sequence identity, and the Hu23 light chain variable region and the Hu32 heavy chain variable region are combined into a new light / heavy chain variable region combination. The experimental results show that the humanized antibodies containing the new light / heavy chain variable region combination all maintain the binding ability to the PD-1 antigen (Table 16).

[0373] Table 13. General formula of Hu32 and Hu23 antibody variable region consensus sequences

[0374]

[0375]

[0376] Table 14. Combinations of Hu32 heavy chain variable regions with Hu23 light chain variable regions

[0377]

[0378] Note: For example, "Hu32a-85" in the table refers to an antibody light / heavy chain variable region combination in which the light chain variable region is Hu23 VL1 (N28T) and the heavy chain variable region is Hu32 VH6, and so on.

[0379] The antibody light / heavy chain variable region combinations referred to in the above table (e.g., Hu32a-85) can be linked to antibody light / heavy chain constant regions, respectively, to form full-length antibodies; in the present disclosure, unless otherwise specified, when a full-length antibody is formed, the light chain variable region is linked to the Kappa chain constant region set forth in SEQ ID NO: 73 to form an antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region set forth in SEQ ID NO: 72 or the IgG4-P heavy chain constant region set forth in SEQ ID NO: 79 to form an antibody heavy chain, and the name of the antibody light / heavy chain variable region combination referred to in the table (e.g., Hu32a-85) is suffixed with ".IgG4AA" to indicate a full-length antibody formed with the IgG4-AA heavy chain constant region, or suffixed with ".IgG4P" to indicate a full-length antibody formed with the IgG4-P heavy chain constant region, for example, "Hu32a-85.IgG4AA" indicates a full-length antibody formed with a heavy chain linked from the Hu32 VH6 heavy chain variable region and the IgG4-AA heavy chain constant region set forth in SEQ ID NO: 72, and a light chain linked from the Hu23 VL1 (N28T) light chain variable region and the Kappa chain constant region set forth in SEQ ID NO: 73. "Hu32a-85.IgG4P" indicates a full-length antibody formed with a heavy chain linked from the Hu32 VH6 heavy chain variable region and the IgG4-P heavy chain constant region set forth in SEQ ID NO: 79, and a light chain linked from the Hu23 VL1 (N28T) light chain variable region and the Kappa chain constant region set forth in SEQ ID NO: 73.

[0380] Table 15. Combinations of Hu23 heavy chain variable regions with Hu32 light chain variable regions

[0381]

[0382]

[0383] Note: For example, "Hu23a-57" in the table refers to the antibody light chain variable region combination of Hu32VL1 and Hu23VH1, and the others are similar.

[0384] The antibody light / heavy chain variable region combinations referred to in the above table (e.g., Hu23a-57) can be respectively linked to the antibody light / heavy chain constant regions to form a full-length antibody; unless otherwise specified in the present disclosure, when forming a full-length antibody, the light chain variable region is linked to the kappa chain constant region shown in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or the IgG4-P heavy chain constant region shown in SEQ ID NO: 79 to form the antibody heavy chain. The name of the antibody light / heavy chain variable region combination in the table (e.g., Hu32a-85) plus the suffix ".IgG4AA" indicates a full-length antibody linked to an IgG4-AA heavy chain constant region, and the suffix ".IgG4P" indicates a full-length antibody linked to an IgG4-P heavy chain constant region. For example, "Hu23a-57.IgG4AA" indicates a combination of the Hu23VH1 heavy chain variable region and the heavy chain constant region shown in SEQ ID NO: A full-length antibody comprises a heavy chain consisting of the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, and a light chain consisting of the Hu32VL1 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73. "Hu23a-57.IgG4P" represents a full-length antibody comprising a heavy chain consisting of the Hu23VH1 heavy chain variable region and the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and a light chain consisting of the Hu32VL1 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73.

[0385] 5. Screening of humanized antibodies

[0386] The affinity of different humanized antibodies was tested by Biacore (see Test Example 3 for the method). The results are shown in Table 16. The results show that different humanized antibodies maintain their binding ability to PD-1, and the affinity of some humanized antibodies is even close to that of their mouse antibodies.

[0387] Table 16. Affinity of Hu23 humanized antibody to human PD-1

[0388]

[0389]

[0390] Example 3. Construction and expression of PD-1 humanized antibody

[0391] The primer PCR was designed to construct each humanized antibody VH / VK gene fragment, and then the homologous recombination was performed with the expression vector pHr (with signal peptide and constant region gene (CH1-Fc / CL) fragment) to construct the full-length antibody expression vector VH-CH1-Fc-pHr / VK-CL-pHr. IgG4-P represents S228P (corresponding to position 108 of the sequence SEQ ID NO: 72 or SEQ ID NO: 79) mutation, IgG4-AA represents F234A (corresponding to position 114 of the sequence SEQ ID NO: 72 or SEQ ID NO: 79), L235A (corresponding to position 115 of the sequence SEQ ID NO: 72 or SEQ ID NO: 79) and S228P (corresponding to position 108 of the sequence SEQ ID NO: 72 or SEQ ID NO: 79) mutations, and the IgG4-AA and IgG4-P antibody forms can be obtained by simply mutating the IgG4 antibody form.

[0392] The IgG4-AA heavy chain constant region sequence is as follows (SEQ ID NO: 72):

[0393]

[0394] The antibody light chain (Kappa chain) constant region sequence is as follows (SEQ ID NO: 73):

[0395]

[0396] The constructed IgG4AA form full-length antibody sequence is exemplarily listed as follows:

[0397] Hu23-11.IgG4AA antibody heavy chain (SEQ ID NO: 74):

[0398]

[0399]

[0400] Hu23-11.IgG4AA light chain (SEQ ID NO: 75):

[0401]

[0402] Hu32a-85.IgG4AA heavy chain (SEQ ID NO: 76):

[0403]

[0404] Hu32a-85.IgG4AA light chain (same as the light chain of Hu23-11.IgG4AA, SEQ ID NO: 75):

[0405]

[0406] Hu33-6.IgG4AA heavy chain (SEQ ID NO: 77):

[0407]

[0408] Hu33-6.IgG4AA light chain (SEQ ID NO: 78):

[0409]

[0410]

[0411] The heavy chain constant region sequence of IgG4-P is as follows (SEQ ID NO: 79):

[0412]

[0413] The constructed IgG4-P format full-length antibody sequence is exemplarily listed as follows:

[0414] Hu23-11.IgG4P antibody heavy chain (SEQ ID NO: 80):

[0415]

[0416] Hu23-11.IgG4P light chain (same as the light chain of Hu23-11.IgG4AA, SEQ ID NO: 75):

[0417]

[0418] Hu32a-85.IgG4P heavy chain (SEQ ID NO: 81):

[0419]

[0420] Hu32a-85.IgG4P light chain (same as the light chain of Hu23-11.IgG4AA, SEQ ID NO: 75):

[0421]

[0422] Hu33-6.IgG4P heavy chain (SEQ ID NO: 82):

[0423]

[0424] Hu33-6.IgG4P light chain (same as the light chain of Hu33-6.IgG4AA, SEQ ID NO: 78):

[0425]

[0426] Test Example

[0427] Test Example 1. Binding of anti-PD-1 antibody to PD-1 ligand and binding blockade ELISA experiment in vitro

[0428] PD-L1 on the surface of tumor cells inhibits the proliferation of T cells by binding to PD-1 on the surface of T cells. Antibodies against PD-1 can block the PD-L1 / PD-1 signaling pathway by binding to PD-1, thereby stimulating the proliferation of T cells. The PD-1 / PD-L1 binding blockade experiment is used to detect the blocking activity of anti-PD-1 antibodies on the signaling pathway.

[0429] In this experiment, after coating the 96-well plate with PD-1-His protein (Cat. # 10377H08H, Sino Biological), the anti-PD-1 antibodies to be tested (including antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see H005-1 antibody in WO2015085847) were added and incubated; later, HRP-labeled goat anti-human IgG (H+L) antibody (Cat. # 109-035-003, Jackson ImmunoResearch) was added and incubated. After washing the plate, the amount of HRP-labeled goat anti-human IgG (H+L) binding was detected, and the EC 50 values of anti-PD-1 antibodies to ligand PD-1 binding were calculated.

[0430] In this experiment, after coating the 96-well plate with PD-1-His protein (Cat. # 10377H08H, Sino Biological), the anti-PD-1 antibodies to be tested (including antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see H005-1 antibody in WO2015085847) were added and incubated; later, HRP-labeled goat anti-human IgG (H+L) antibody (Cat. # 109-035-003, Jackson ImmunoResearch) was added and incubated. After washing the plate, the amount of HRP-labeled goat anti-human IgG (H+L) binding was detected, and the EC 50 values of anti-PD-1 antibodies to ligand PD-1 binding were calculated.

[0431] PD-1-Fc was diluted to 1 μg / mL with pH 9.6 CB buffer (1.59 g Na2CO3 and 2.93 g NaHCO3 were dissolved in 1 L distilled water) and added to 96-well plates at a volume of 100 μL / well, and placed at 4°C for 16-20 h. The PBS buffer in the 96-well plates was aspirated, and the plates were washed once with PBST (pH 7.4 PBS containing 0.05% tween 20) buffer, and 120 μL / well of PBST / 1% milk was added for blocking at room temperature for 1 h. The blocking solution was removed, and the plates were washed once with PBST buffer, and 90 μL of the test anti-PD-1 antibody diluted to the appropriate concentration with sample diluent (pH 7.4 PBS containing 5% BSA, 0.05% Tween 20) was added, and pre-incubated at 4°C for 1 h. Biotin-labeled PD-L1 / PD-L2 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) (10 μg / mL) was added at a 10x concentration at a volume of 10 μL / well, and after shaking and mixing on a shaker, incubated at 37°C for 1 h. The reaction system was removed, and the plates were washed 6 times with PBST, and 100 μL / well of Streptavidin-Peroxidase Polymer (streptavidin-peroxidase polymer) diluted 1:400 with PBST buffer was added, and shaken and incubated at room temperature for 50 min. The plates were washed 6 times with PBST, and 100 μL / well of TMB was added, and incubated at room temperature for 5-10 min. The reaction was terminated by adding 100 μL / well of 1 M H2SO4. The absorbance value was read at 450 nm with a microplate reader, and the IC50 value of the anti-PD-1 antibody for blocking the binding of the ligand PD-L1 / PD-L2 was calculated. 50 The data are shown in Table 17 below.

[0432] Table 17. Anti-PD-1 antibodies of the disclosure and PD-1 binding and blocking of the binding of the ligand PD-L1 / PD-L2 ELISA

[0433]

[0434] The exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA, and Hu33-6.IgG4AA can all effectively block the binding of PD-1 to PD-L1 / PD-L2, and their blocking activity is similar to that of the positive control antibody.

[0435] Test Example 2. Ligand blocking test of exemplary antibodies

[0436] The blocking effect of the antibodies on the binding of PD-1 to PD-L1 was investigated. The experimental process is briefly described as follows:

[0437] Jurkat / PD-1 cells (Jurkat cells stably transfected with PD-1) were counted and plated in a 96-well plate at a ratio of CHOK1 / PD-L1 cells (Promega) (90 μL / well) and 10 μL / well of the diluted antibodies (antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA, and Hu33-6.IgG4AA, positive control antibody: H005-1), negative control IgG4 protein, antibody gradient dilution concentrations of 0.3 mg / mL, 3 mg / mL, 30 mg / mL) were added, and the plate was incubated at 37 °C in a 5% CO2 incubator for 5 hours. The cell culture plate was removed and placed at room temperature for 5 minutes, and then 50 μL Bio-Glo® TM Reagent was added to each well, and the plate was incubated at room temperature for 5 minutes, and then read. The experimental results are shown in Figure 6. Figure 2 .

[0438] The results show that the exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA, and Hu33-6.IgG4AA in the present disclosure can effectively block the binding of PD-1 to PD-L1.

[0439] Test Example 3. BIAcore antibody affinity experiment of exemplary antibodies

[0440] The Protein A biosensor chip (Cat. #29127556, GE) was used to capture IgG, and the human PD-1 antigen (Cat. #10377H08H, Sino Biological) and Cyno PD-1 antigen (purchased from Sino Biological) were flowed through the chip surface. The Biacore T200 instrument was used to detect the binding and dissociation curves of the PD-1 antibody and the antigen PD-1 in real time. After each experimental cycle was completed, the biosensor chip was washed and regenerated with a 10 mM Glycine-HCl pH 1.5 buffer. The experimental buffer system was 1x HBS-EP buffer solution (Cat #BR-1001-88, GE). After the experiment, the data was fitted with the (1:1) Langmuir model using the GE Biacore T200 Evaluation version 3.0 software to obtain the affinity values, and the results are shown in Table 18.

[0441] Table 18. Affinity of anti-PD-1 antibodies to human PD-1 and monkey PD-1

[0442]

[0443] Results show that the exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA of the present disclosure are all capable of binding to human PD-1 and monkey PD-1.

[0444] Test Example 4. Effect of antibodies on cell IFNγ secretion in PBMC-T lymphocyte activation experiment

[0445] To study the effect of anti-PD-1 antibodies on the function of human primary T lymphocytes, human peripheral blood mononuclear cells (PBMC) were collected and purified, and after in vitro stimulation with tuberculin (TB) for 5 days, the cytokine IFNγ secretion level was detected. The experimental process is simply described as follows:

[0446] Fresh blood was subjected to density gradient centrifugation (Stem Cell Technologies) using Ficoll-Hypaque (17-5442-02, GE) to obtain PBMC, which were cultured in RPMI 1640 (SH30809.01, GE) medium supplemented with 10% (v / v) FBS (10099-141, Gibco) at 37°C under 5% CO2 conditions.

[0447] The freshly isolated and purified PBMC were adjusted to a density of 2×10 6 mL of cell suspension was added with 40 μL of tuberculin (97-8800, Synbiotics), and cultured in a 37°C, 5% CO2 incubator for 5 days. On day 5, the cultured cells were collected by centrifugation and resuspended in fresh RPMI 1640 medium to a density of 1.1×10 6 mL. At the same time, gradient-diluted antibody samples (including antibodies of the present disclosure: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody H005-1, and negative control IgG4 protein, with antibody gradient dilution concentrations of 0.3 mg / mL, 3 mg / mL, and 30 mg / mL) were diluted with PBS (B320, Shanghai Yuenpure Biotech Co., Ltd.) at 10 μL per well. The cell culture plate was incubated in a 37°C, 5% CO2 incubator for 3 days. The cell culture plate was removed, and the cell culture supernatant was collected by centrifugation (4000 rpm, 10 min). The level of IFN-γ was detected by ELISA (human IFN-γ detection kit (EHC102g.96, Xinboseng)). For specific operations, refer to the reagent instruction manual.

[0448] The results are shown in Table 1. Figure 3 The results show that the anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA, and Hu33-6.IgG4AA of the present disclosure can effectively activate the secretion of IFN-γ.

[0449] Test Example 5. Effect of anti-PD-1 antibodies in transgenic PD-1 mouse colon cancer model MC38

[0450] MC38 cells 5×10 5 were inoculated subcutaneously in the right flank of 90 hPD-1 TG mice (BIOSEGEN) at 5×10 3 The tumor volume, animal weight were monitored twice a week and the data were recorded. When the tumor volume exceeded 2000 mm 长 or most of the tumors were ulcerated or the body weight decreased by 20%, the tumor-bearing animals were euthanized as the end point of the experiment.

[0451] Tumor volume (TV) = 1 / 2 x L 短 2

[0452] Tumor proliferation rate (T / C%) = (T-T0) / (C-C0) x 100%

[0453] Tumor inhibition rate (TGI%) = 1-T / C%

[0454] Wherein T, T0 represent the tumor volume of the antibody administration group at the end of the test and at the beginning of the test, respectively, and C, C0 represent the tumor volume of the blank control group at the end of the test and at the beginning of the test, respectively.

[0455] The results are shown in Table 19 and the attached Figure 4The results show that, compared with the blank control, the antibodies of the present disclosure can significantly inhibit the growth of mouse colon cancer MC38 transplanted tumors, and the highest tumor inhibition rate is the Hu32a-85.IgG4AA-3mpk group, with a tumor inhibition rate of 77.64% at the last measurement. When the administration frequency is three times a week for three times, the results show that the tumor inhibition rates of the antibodies of the present disclosure are significantly better than those of the positive control antibody H005-1 when detected on the seventh day; thereafter, the administration frequency is reduced to once a week, and after two administrations (Day 21), the efficacy of the antibodies of the present disclosure gradually diverges, and shows a dose-dependent manner, wherein Hu32a-85.IgG4AA is significantly better than H005-1 at the same dose (p<0.05). Moreover, the tumor-bearing mice can well tolerate the anti-PD-1 antibodies, and the body weight steadily increases during the entire administration process, without obvious drug-induced body weight loss and other symptoms.

[0456] Table 19. Effect of anti-PD-1 antibodies on the tumor inhibition rate of mouse colon cancer MC38 (mm 3 )

[0457]

[0458] Test Example 6. Effect of anti-PD-1 antibodies in a transgenic PD-1 mouse colon cancer model MC38

[0459] The transgenic PD-1 mice were derived from the fifth generation mice of the purchased transgenic PD-1 mice (ISIS INNOVATION LIMITED, University Offices, Wellington Square, Oxford OX1 2JD, England) bred at Cephrim Biosciences, Inc. MC38 cells were inoculated subcutaneously at 5x10 5 mm3, the animals with excessive body weight, tumor size, and small tumor size were removed, and the tumor-bearing mice were randomly divided into 5 groups (8 mice per group) according to the tumor size: negative control hIgG control 30mpk, H005-1 10mpk, H005-1 30mpk, Hu33-6.IgG4AA 10mpk, Hu33-6.IgG4AA 30mpk. The grouping administration date was set as Day 0. After grouping, each drug was administered intraperitoneally, and the administration cycle was 22 days, once every two days, for a total of 11 times. The tumor volume was measured twice a week, the body weight was measured, and the data was recorded. The body weight and tumor volume of the animals in each group were represented by the mean ± standard deviation (Mean ± SEM), and were plotted using Graphpad Prism 5 and Excel software, and were statistically analyzed using student t test.

[0460] Tumor volume (TV) = 0.5236 × L 长 ×L 短 2

[0461] Tumor proliferation rate T / C% = (T-T0) / (C-C0) × 100%

[0462] Tumor inhibition rate %TGI=1-T / C%

[0463] T and T0 represent the tumor volumes of the antibody administration group at the end and the beginning of the experiment, respectively; C and C0 represent the tumor volumes of the blank control group at the end and the beginning of the experiment, respectively.

[0464] The test results are shown in Table 20 and Appendix Hu23-11. IgG4 AA As shown, the test results show that compared with the control group, the antibodies disclosed herein can significantly inhibit the growth of mouse colon cancer MC38 xenograft tumors, among which the highest tumor inhibition rate was in the Hu33-6.IgG4AA 30mpk group, with a tumor inhibition rate of 80.4% when measured on day 20. In the low-dose group (10mpk), the efficacy of Hu33-6.IgG4AA-10mpk was better than that of the positive control H005-1-10mpk.

[0465] Table 20. Effect of anti-PD-1 antibodies on the tumor volume of mouse colon cancer MC38

[0466]

[0467] Note: The unit of the average tumor volume of each group in the table is: mm 3 .

[0468] Test Example 7. Pharmacokinetic Study of Anti-PD-1 Antibody in Cynomolgus Monkeys

[0469] Six male cynomolgus macaques, aged 2–5 years and weighing 2–5 kg, were purchased from Guangdong Qianxian Biotechnology Co., Ltd. (license number: SCXK(粤)2015-0037, animal qualification certificate number: 44613900000219).

[0470] Rearing environment: Room temperature controlled at 18-26°C, relative humidity at 40%-70%, with a 12-hour light-dark cycle. Except in cases where fasting is required, feed and water are available in unlimited quantities.

[0471] Animals were weighed before dosing, and the body weight was between 2.81-3.52 kg. The animals were dosed with 1 mg / kg (1 mpk) by subcutaneous intravenous infusion using a syringe pump in the front or back limbs, and the dosing speed was 0.1 mL / kg / min, and the dosing time was about 30 min. The animals were collected with whole blood from the back limbs at the following time points: before dosing, 5 min, 0.25 h, 0.5 h (immediately after dosing), 1 h, 2 h, 4 h, 8 h, 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 10 d, 13 d, 14 d, 21 d, and 28 d after the start of intravenous infusion, and the serum was separated. Among them, about 2 mL of whole blood was collected before dosing, 14 d, 21 d, and 28 d after the start of intravenous infusion, and about 1 mL of whole blood was collected at the other blood collection points. The serum drug concentration was detected by ELISA, and the PK analysis was performed, and the results are shown in Table 21.

[0472] Table 21. Pharmacokinetics of humanized anti-PD-1 antibodies in cynomolgus monkeys

[0473] Hu33-6. IgG4 AA t1 / 2 (day) Cmax (pg / mL) 5.5±0.7 4.6±1.3 AUC (h*pg / mL) 23.75±2.29 21.47±2.13 CL (mL / day / kg) 2775±241 2319±518 Vz (mL / kg) 8.7±0.7 10.7±2.3 Figure 5 69±3.6 67.9±3.4

[0474] The results show that the pharmacokinetic activity of Hu23-11.IgG4AA and Hu33-6.IgG4AA is good.

[0475] The following is an exemplary test for preparing a stable formulation of an anti-PD-1 antibody. The anti-PD-1 antibody used in the following formulation implementation is Hu23-11.IgG4AA described above, and the equipment used in the preparation process and the calculation method of the results are as follows:

[0476] SEC molecular exclusion chromatography: an analytical method for separating solutes according to the relative relationship between the pore size of the gel pores and the size of the polymer sample molecules. SEC monomer content percentage = Amonomer / Atotal*100% (Amonomer is the peak area of the main monomer peak in the sample, and Atotal is the sum of all peak areas). SEC determination instrument: Agilent 1260; column: waters, XBrige SEC (300 x 7.8 mm 3.5 μm).

[0477] CE capillary gel electrophoresis: a method of electrophoresis in which the gel is moved into a capillary as a support medium and separated under a certain voltage according to the size of the sample molecular weight. Non-reduced CE (NR-CE) purity percentage = Apeak / Atotal*100% (Apeak is the peak area of the main peak in the sample, and Atotal is the sum of all peak areas. CE determination instrument: Beckman, model plus800.

[0478] iCIEF imaging capillary isoelectric focusing electrophoresis (iCE): a technique that separates proteins according to their isoelectric point, pi. iCIEF neutral peak content percentage = neutral peak area / total area * 100% (total area is the sum of the acid peak, neutral peak and basic peak areas). Instrument used for iCIEF determination: simple protein, model muarice.

[0479] Osmotic pressure: Osmotic pressure is determined by the freezing point method, which is based on the proportional relationship between the freezing point depression and the molar concentration of the solution. A high-sensitivity temperature sensor is used to measure the freezing point of the solution, which is converted into osmotic pressure by electric quantity. Instrument manufacturer: Loser, model OM815.

[0480] Test Example 8: Buffer system and pH screening of anti-PD-1 antibody formulation

[0481] Using the following buffers, anti-PD-1 antibody (Hu23-11.IgG4AA) formulations containing 80 mg / mL sucrose and 0.6 mg / mL polysorbate 80 (PS80) with a protein concentration of 100 mg / mL were prepared, wherein the buffers were as follows:

[0482] 1) 10 mM sodium acetate (abbreviated as: AA), pH 5.0;

[0483] 2) 10 mM sodium acetate, pH 5.2;

[0484] 3) 10 mM sodium acetate, pH 5.5;

[0485] 4) 10 mM sodium acetate, pH 5.7;

[0486] 5) 10 mM sodium succinate (abbreviated as: SA), pH 5.2;

[0487] 6) 10 mM sodium citrate (abbreviated as: CA), pH 5.2;

[0488] The prepared formulations were filtered, filled, plugged, and capped. The stability of the samples under forced degradation conditions (40°C M1, i.e., 40°C high temperature for 1 month) and accelerated conditions (25°C M6, i.e., 25°C temperature for 6 months) was investigated, and the appearance, SEC and non-reduced CE-SDS were used as evaluation indexes to investigate the stability of the formulations. The experimental results are shown in Table 22.

[0489] Under the forced degradation conditions of 40°C M1, the appearance of the protein formulations of the AA and SA buffer systems was better than that of the CA system; the purity of the protein formulations of the AA buffer system at pH 5.2-5.7 was better than that of the AA buffer system at pH 5.0 and the SA buffer system at pH 5.2; under the accelerated conditions of 25°C M6, there was no significant difference between the appearance groups.

[0490] Table 22. Results of pH and buffer system screening experiments

[0491]

[0492] Note: D0 means the beginning of the experiment, "M" means month, for example, M1 means one month.

[0493] In addition, for another batch of anti-PD-1 antibody (Hu23-11.IgG4AA), the following buffers were used to prepare anti-PD-1 antibody (Hu23-11.IgG4AA) formulations containing 80 mg / mL sucrose and 0.6 mg / mL polysorbate 80 (PS80) with a protein concentration of 100 mg / mL, wherein the buffers are as follows:

[0494] 1) 10 mM sodium acetate, pH 5.2;

[0495] 2) 10 mM sodium succinate, pH 5.2;

[0496] 3) 10 mM histidine acetate (abbreviation: His-AA), pH 5.2;

[0497] The prepared formulations were filtered, filled, plugged, and capped. The samples were investigated for stability under accelerated conditions (25°C M6, i.e., 6 months at 25°C), and the SEC and iCIEF indicators of the formulations were investigated to study the stability of the formulations. The experimental results are shown in Table 23.

[0498] Under accelerated conditions (25°C M6), the SEC / iCIEF data of pH 5.2 AA and pH 5.2 His-AA were higher than that of the pH 5.2 SA group, and the iCIEF of the pH 5.2 AA buffer system was slightly higher than that of the pH 5.2 His-AA buffer system. In consideration of the above, the preferred buffer system of the formulation is acetate and histidine salt buffer system, and the preferred buffer system is AA or His-AA buffer system.

[0499] Table 23. Results of pH and buffer system screening experiments

[0500]

[0501] Note: "D" in the table means day, D0 means the beginning of the experiment, "M" means month, for example, M1 means one month.

[0502] Test Example 9: Ion strength screening of anti-PD-1 antibody formulation buffer system

[0503] The anti-PD-1 antibody (Hu23-11.IgG4AA) formulation with 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and a protein concentration of 120 mg / mL was prepared using sodium acetate buffer with an ionic strength of 10 mM and 30 mM at pH 5.2, respectively. The excipient formulation:

[0504] 1) 10 mM pH 5.2 AA, 0.6 mg / mL PS80, and 80 mg / mL sucrose;

[0505] 2) 30 mM pH 5.2 AA, 0.6 mg / mL PS80, and 80 mg / mL sucrose;

[0506] The prepared formulation was filtered, filled, stoppered, and crimped. The stability of the samples under forced degradation conditions (40°C M1) was investigated, and the SEC was used as an evaluation index to investigate the stability of the formulation. The experimental results are shown in Table 24.

[0507] The experimental data showed that under the forced degradation conditions of 40°C M1, the SEC data of the 10 mM ionic strength group was slightly higher than that of the 30 mM ionic strength group, and therefore the ionic strength of the pH 5.2 sodium acetate buffer system was preferably 10 mM.

[0508] Table 24. Experimental results of ionic strength screening

[0509]

[0510] Note: “D” in the table represents days, and DO represents the time when the experiment started. “M” represents months, for example, M1 represents one month.

[0511] Test Example 10: Surfactant screening in anti-PD-1 antibody formulation

[0512] The anti-PD-1 antibody (Hu23-11.IgG4AA) formulation with the following 1)-5) different concentrations of Tween 80 was prepared in 10 mM sodium acetate buffer at pH 5.2. The protein concentration was 100 mg / mL, and the other excipients were as follows:

[0513] 1) 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80 (PS80);

[0514] 2) 80 mg / mL sucrose, 0.4 mg / mL PS80;

[0515] 3) 80 mg / mL sucrose, 0.6 mg / mL PS80;

[0516] 4) 80 mg / mL sucrose, 0.8 mg / mL PS80;

[0517] 5) 80 mg / mL sucrose, 0.6 mg / mL polysorbate 20 (PS20).

[0518] The finished formulation was filtered, filled, stoppered, and crimped. The samples were subjected to shake (25 °C, 300 rpm, 10 days) and long-term stability (2-8 °C for 6 months) experiments, with appearance, SEC, and non-reduced CE-SDS as evaluation indexes. The experimental results are shown in Table 25.

[0519] The experimental results show that, under the shake D10 condition, the appearance of the 0.6 mg / mL PS80, 0.8 mg / mL PS80, and 0.6 mg / mL PS20 groups is better than that of the 0.2 mg / mL PS80 and 0.4 mg / mL PS80 groups, in which a large number of particles appear; when placed at 2-8 °C for 6 months, the 0.6 mg / mL PS80 and 0.8 mg / mL PS80 groups are better than the 0.6 mg / mL PS20 group in terms of appearance, in which turbidity appears, and there is no significant difference in purity between the groups. Therefore, the surfactant is selected to be polysorbate 80, and the concentration is preferably 0.6 mg / mL.

[0520] Table 25. Experimental results of surfactant screening in the formulation

[0521]

[0522] Note: “D” in the table represents days, for example, D10 represents 10 days; D0 represents the time when the experiment starts, and “M” represents months, for example, M6 represents six months.

[0523] Test Example 11: Screening of Osmotic Pressure Regulators in Anti-PD-1 Antibody Formulations

[0524] Anti-PD-1 antibody (Hu23-11.IgG4AA) formulations with a protein concentration of 100 mg / mL were prepared in 10 mM sodium acetate buffer at pH 5.2 containing the following 1)-7) different types of excipients:

[0525] 1) 0.6 mg / mL polysorbate 80 (PS80);

[0526] 2) 80 mg / mL sucrose, 0.6 mg / mL PS80;

[0527] 3) 80 mg / mL trehalose, 0.6 mg / mL PS80;

[0528] 4) 50 mg / mL sorbitol, 0.6 mg / mL PS80;

[0529] 5) 100 mM arginine, 0.6 mg / mL PS80;

[0530] 6) 100 mM glycine, 0.6 mg / mL PS80;

[0531] 7) 100 mM NaCl, 0.6 mg / mL PS80;

[0532] The prepared formulations were filtered, filled, stoppered, and crimped. The stability of the samples under forced degradation conditions (40 °C D22 (22 days at 40 °C)) was investigated, and the appearance, SEC, and non-reduced CE-SDS were used as evaluation indexes to investigate the stability of the formulations. The experimental results are shown in Table 26.

[0533] Under the forced degradation conditions of 40 °C D22, there was no significant difference in appearance among the groups; from the data of purity, the SEC and non-reduced CE-SDS of the 80 mg / mL sucrose, 80 mg / mL trehalose, and 50 mg / mL sorbitol groups were slightly higher than those of the other groups, and there was no significant difference in purity among the three groups of formulations. When the administration route is subcutaneous injection, the osmotic pressure of the formulation is controlled at 280-320 mOsm, and therefore, the sucrose content is preferably 70-90 mg / mL. The osmotic pressure of the formulation containing 80 mg / mL sucrose is about 300 mOsm, and the sucrose content is preferably 80 mg / mL.

[0534] Table 26. Experimental results of osmotic pressure screening of formulations

[0535]

[0536] Note: "D" in the table represents days, for example, D22 represents 22 days; D0 represents the start of the experiment.

[0537] Test Example 12: Protein concentration screening of anti-PD-1 antibody formulations

[0538] Using a buffer system of 10 mM sodium acetate salt at pH 5.2, an anti-PD-1 antibody (Hu23-11.IgG4AA) formulation containing 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80 (PS80), and having a protein concentration of 100 mg / mL or 120 mg / mL was prepared.

[0539] 1) 100 mg / mL protein concentration, 0.6 mg / mL PS80, and 80 mg / mL sucrose;

[0540] 2) 120 mg / mL protein concentration, 0.6 mg / mL PS80, and 80 mg / mL sucrose;

[0541] Each formulation was filtered, filled, stoppered, and crimped. The stability of the samples under forced degradation conditions (40 °C M1) was investigated, and the appearance, SEC, non-reduced CE-SDS, and ICE were used as evaluation indexes to investigate the stability of the formulations. The experimental results are shown in Table 27.

[0542] The experimental data showed that there was no significant difference in appearance and purity between the 100 mg / mL and 120 mg / mL formulation groups under the 40°C M1 forced degradation condition.

[0543] Table 27. Comparison of experimental results of different concentrations of formulations

[0544]

[0545] Note: "D" in the table represents days, D0 represents the time when the experiment starts, and "M" represents months, for example, M1 represents one month.

[0546] Test Example 13: Anti-PD-1 antibody formulation stability experiment

[0547] An anti-PD-1 antibody (Hu23-11.IgG4AA) formulation containing a protein concentration of 120 mg / mL, 10 mM sodium acetate salt pH 5.2, 80 mg / mL sucrose, and 0.6 mg / mL PS80 was prepared.

[0548] The prepared formulation was filtered, filled, plugged, and capped. The stability of the sample was investigated under forced degradation conditions (40°C M1), freeze-thaw 5 times, and shaking D7 (25°C, 300 rpm), and the stability of the formulation was investigated with appearance, SEC, non-reduced CE-SDS, and IEC as evaluation indexes. The experimental results are shown in Table 28.

[0549] The experimental results showed that the final formulation maintained clarity from the appearance under each forced degradation condition; from the purity data, there was no significant change under freeze-thaw 5 times or shaking for 7 days (25°C, 300 rpm), a decrease of 1.9% in SEC, a decrease of 2.5% in NR-CE, and a decrease of 14.8% in IEC under the 40°C M1 forced degradation condition compared to D0, and the formulation had good stability.

[0550] Table 28. Formulation stability experiment results

[0551]

[0552] Note: "D" in the table represents days, D0 represents the time when the experiment starts, and "M" represents months, for example, M1 represents one month.

[0553] In addition, the stability of the preparation under accelerated degradation conditions of 25°C M6 (6 months at 25°C) and 4°C M6 (6 months at 4°C) was also observed, and the experimental results are shown in Table 29. The experimental results show that the preparation of the present disclosure has good stability under the long-term condition of 4°C M6, and the preparation of the present disclosure still has good stability under the accelerated degradation condition of 25°C M6, and the SEC of the preparation only decreases by 2.2%, the non-reduced CE-SDS only decreases by 2.7%, and the IEC decreases by 9.3% compared with D0.

[0554] Table 29. Experimental results of preparation stability

[0555]

[0556] Test Example 14: Anti-PD-1 antibody preparation prescription optimization experiment

[0557] The DOE experiment was designed with the ion strength, pH value and protein concentration of sodium acetate salt as variables. The DOE experiment factors and levels were set as ion strength 10-30 mM, pH 4.7-5.7, and anti-PD-1 antibody (Hu23-11.IgG4AA) concentration 90-150 mg / mL. A series of prescriptions were designed (see Table 30). Through forced degradation experiment under high temperature condition of 40°C for one month, appearance, SEC and IEC were used as evaluation indexes, and the results were statistically analyzed by least square method. The results are shown in Table 31 and Group .

[0558] The results show that under the condition of 40°C high temperature for one month, the appearance of each prescription is clear, the purity item SEC decreases by 1.1%-2.7%, and the iCIEF decreases by 5.6%-8.5%, which is within the acceptable range of forced degradation condition, and the difference between groups is within the detection error range of the instrument. Therefore, the anti-PD-1 antibody sample has good stability within the range of protein concentration 90-150 mg / mL, ion strength 10-30 mM and pH 4.7-5.7. The data is fitted, and it is observed that the purity item is in a safer range at low ion strength, so the protein concentration 120 mg / mL, ion strength 10 mM and pH 5.2 can be preferred.

[0559] Table 30. Prescription design of DOE prescription screening experiment

[0560] pH Protein Concentration (mg / mL) Ionic Strength (mM) Hu23-11. IgG4 AA Hu33-6. IgG4 AA t1 / 2 (day) Cmax (pg / mL) AUC (h*pg / mL) CL (mL / day / kg) Vz (mL / kg) Figure 5 Group pH Protein Concentration (mg / mL) Ionic Strength (mM) Hu23-11. IgG4 AA Hu33-6. IgG4 AA t1 / 2 (day) Cmax (pg / mL) AUC (h*pg / mL) CL (mL / day / kg) Vz (mL / kg) Figure 5 Group pH Protein Concentration (mg / mL) Ionic Strength (mM) Hu23-11. IgG4 AA Hu33-6. IgG4 AA t1 / 2 (day) Cmax (pg / mL) AUC (h*pg / mL) CL (mL / day / kg) Vz (mL / kg) Figure 5 Group pH Protein Concentration (mg / mL) Ionic Strength (mM) Hu23-11. IgG4 AA Hu33-6. IgG4 AA t1 / 2 (day) Cmax (pg / mL) AUC (h*pg / mL) CL (mL / day / kg) Vz (mL / kg) Figure 5 Group pH Protein Concentration (mg / mL) Ionic Strength (mM) Hu23-11. IgG4 AA Hu33-6. 1 5.7 150 20 2 4.7 120 21.9 3 5.2 120 10 4 4.7 90 10 5 5.2 120 20 6 5.2 90 20 7 5.7 150 10 8 4.7 90 30 9 4.7 150 10 10 5.2 150 30 11 5.7 90 10 12 5.7 107.7 30

[0561] Note: All prescriptions add 80 mg / mL sucrose and 0.6 mg / mL PS80.

[0562] Table 31. Results of DOE screening experiment

[0563]

[0564] Note: In the table, "D" means day, DO means at the beginning of the experiment, "M" means month, for example, M1 means one month; N means not applicable in this table; SEC monomer decrease (%) = the difference between the SEC monomer (%) of the preparation at 40℃ M1 and the SEC monomer (%) of the preparation at DO; iCIEF neutral peak decrease (%) = the difference between the iCIEF neutral peak (%) of the preparation at 40℃ M1 and the iCIEF neutral peak (%) of the preparation at DO.

[0565] Test Example 15: Lyophilization of Anti-PD-1 Antibody Formulation

[0566] An anti-PD-1 antibody (Hu23-11.IgG4AA) formulation containing 100 mg / mL antibody, 80 mg / mL sucrose, 0.6 mg / mL PS80 was prepared in a 10 mM AA pH 5.2 buffer, and the formulation sample was lyophilized, with a pre-freeze, primary drying and secondary drying (parameters see Table 32). After the end of the lyophilization process, the vacuum was plugged. The reconstituted sample was compared before and after lyophilization. The results showed that the reconstituted solution could maintain good performance of the liquid preparation.

[0567] Table 32. Lyophilization process

[0568]

[0569]

[0570] While the foregoing application has been described in some detail for purposes of clarity and understanding, it will be appreciated that certain changes and modifications can be practiced within the scope of the appended claims. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference. SEQUENCE LISTING <110> Jiangsu Hengrui Medicine Co., Ltd. Shanghai Hengrui Pharmaceutical Co., Ltd. <120> Anti-PD-1 antibody pharmaceutical composition and use thereof <130> 721086CPT <150> CN202010755954.9 <151> 2020-07-31 <150> CN202110831575.8 <151> 2021-07-22 <160> 82 <170> SIPOSequenceListing 1.0 <210> 1 <211> 401 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Human PD-1-IgGl Fc sequence <400> 1 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn 20 25 30 Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn 35 40 45 Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu 50 55 60 Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala 65 70 75 80 Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val 85 90 95 Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala 100 105 110 Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala 115 120 125 Pro Lys Ala Gin He Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr 130 135 140 Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg 145 150 155 160 Pro Ala Gly Gin Phe Gin Thr Leu Val Glu Pro Lys Ser Ser Asp Lys 165 170 175 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 180 185 190 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200 205 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 210 215 220 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 225 230 235 240 Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 245 250 255 Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 260 265 270 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys 275 280 285 Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 290 295 300 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 305 310 315 320 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 325 330 335 Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 340 345 350 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 355 360 365 Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 370 375 380 Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 385 390 395 400 Lys <210> 2 <211> 186 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Human PD-1 -his sequence <400> 2 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn 20 25 30 Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn 35 40 45 Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu 50 55 60 Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala 65 70 75 80 Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val 85 90 95 Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala 100 105 110 Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala 115 120 125 Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr 130 135 140 Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg 145 150 155 160 Pro Ala Gly Gln Phe Gln Thr Leu Val Gly Ser Ser Asp Tyr Lys Asp 165 170 175 Asp Asp Asp Lys His His His His His His 180 185 <210> 3 <211> 288 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Transfected cell PD-1 antigen sequence <400> 3 Met Gin He Pro Gin Ala Pro Trp Pro Val Val Trp Ala Val Leu Gin 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gin Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gin Pro Gly Gin Asp Cys Arg Phe Arg 85 90 95 Val Thr Gin Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 4 <211> 125 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> Heavy chain variable region sequence of M23 <400> 4 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Thr Pro Ile His Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Thr Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr His Cys 85 90 95 Thr Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe 100 105 110 Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 5 <211> 112 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> Light chain variable region sequence of M23 <400> 5 Asp Gly Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp His Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 6 <211> 120 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> M32 heavy chain variable region sequence <400> 6 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr 20 25 30 Glu Ile His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Thr 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 7 <211> 112 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> Light chain variable region sequence of M32 <400> 7 Asp Val Leu Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gin Ala Ser lie Ser Cys Arg Ser Ser Gin Ser lie Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Lys Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly lie Tyr Tyr Cys Phe Gin Gly 85 90 95 Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Leu Glu lie Lys 100 105 110 <210> 8 <211> 5 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HCDR1 sequence of M23 <400> 8 Asp Tyr Glu Met His 1 5 <210> 9 <211> 17 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HCDR2 sequence of M23 <400> 9 Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 10 <211> 16 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HCDR3 sequence of M23 <400> 10 Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe Asp Val 1 5 10 15 <210> 11 <211> 16 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> LCDR1 sequence of M23 <400> 11 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu Glu 1 5 10 15 <210> 12 <211> 7 <212> PRT <213> Mouse sequence (Mus musculus) <220> <221> DOMAIN <223> LCDR2 sequence of M23 / M32 <400> 12 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 13 <211> 9 <212> PRT <213> Mouse sequence (Mus musculus) <220> <221> DOMAIN <223> LCDR3 sequence of M23 <400> 13 Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 <210> 14 <211> 5 <212> PRT <213> Mouse sequence (Mus musculus) <220> <221> DOMAIN <223> HCDR1 sequence of M32 <400> 14 Asp Tyr Glu Ile His 1 5 <210> 15 <211> 17 <212> PRT <213> Mouse sequence (Mus musculus) <220> <221> DOMAIN <223> HCDR2 sequence of M32 <400> 15 Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 16 <211> 11 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HCDR3 sequence of M32 <400> 16 Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val 1 5 10 <210> 17 <211> 16 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> LCDR1 sequence of M32 <400> 17 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 18 <211> 9 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> LCDR3 sequence of M32 <400> 18 Phe Gln Gly Ser His Val Pro Tyr Ala 1 5 <210> 19 <211> 118 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> Heavy chain variable region sequence of M33 <400> 19 Lys Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Thr Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 20 <211> 107 <212> PRT <213> 鼠源序列(Mus musculus) <220> <221> DOMAIN <223> M33的轻链可变区序列 <400> 20 Asp Ile GIn Met Thr GIn Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser GIn Asp Ile Asn Asn Phe 20 25 30 Leu Asn Trp Tyr GIn GIn Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu GIn GIn 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys GIn GIn Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu GIn Ile Lys 100 105 <210> 21 <211> 5 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HCDR1 sequence of M33 <400> 21 Ser Tyr Ala Met Ser 1 5 <210> 22 <211> 17 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HCDR2 sequence of M33 <400> 22 Thr lie Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gin Asp Asn Val Gin 1 5 10 15 Gly <210> 23 <211> 9 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HCDR3 sequence of M33 <400> 23 Pro Tyr Gly His Gly Tyr Phe Asp Val 1 5 <210> 24 <211> 11 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> LCDR1 sequence of M33 <400> 24 Arg Ala Ser Gin Asp lie Asn Asn Phe Leu Asn 1 5 10 <210> 25 <211> 7 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> LCDR2 sequence of M33 <400> 25 Tyr Thr Ser Ser Leu His Ser 1 5 <210> 26 <211> 9 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> LCDR3 sequence of M33 <400> 26 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 27 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23VH1 / Hu23VH-CDR grafted sequence <400> 27 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 28 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL1 / Hu23 VL-CDR grafted sequence <400> 28 Asp Ile Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 29 <211> 112 <212> PRT <213> 人工序列(Artificial Sequence) <220> <221> DOMAIN <223> Hu23VL2序列 <400> 29 Asp Gly Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 30 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23VH2 sequence <400> 30 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gin Lys Phe 50 55 60 Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 31 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23VH3 sequence <400> 31 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gin Lys Phe 50 55 60 Lys Asp Arg Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 32 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23VH4 sequence <400> 32 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gin Lys Phe 50 55 60 Lys Asp Arg Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu32VH1 / Hu32VH-CDR grafted sequence <400> 33 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr 20 25 30 Glu He His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly He Val Tyr Asn Gin Lys Phe 50 55 60 Lys Gly Arg Val Thr He Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gin 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 34 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu32VL1 / Hu32VL-CDR grafted sequence <400> 34 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 35 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu32VL2 sequence <400> 35 Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser lie Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gin Gly 85 90 95 Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu lie Lys 100 105 110 <210> 36 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu32 VH2 sequence <400> 36 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Glu lie His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly lie Val Tyr Asn Gin Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gin 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 37 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu32 VH3 sequence <400> 37 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Ser Asp Tyr 20 25 30 Glu lie His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly lie Val Tyr Asn Gin Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gin 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 38 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu32VH4 sequence <400> 38 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Glu He His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp He 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly He Val Tyr Asn Gin Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gin 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 39 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu32VH5 sequence <400> 39 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr 20 25 30 Glu He His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp He 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly He Val Tyr Asn Gin Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gin 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 40 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu32VH6 sequence <400> 40 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr 20 25 30 Glu lie His Trp Val Lys Gin Ala Pro Gly His Gly Leu Glu Trp lie 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly lie Val Tyr Asn Gin Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gin 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 41 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu33VH1 / Hu33VH-CDR grafted sequence <400> 41 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr He Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gin Asp Asn Val 50 55 60 Gln Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gin Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 42 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu33 VL1 / Hu33 VL-CDR grafted sequence <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 43 <211> 107 <212> PRT <213> 人工序列(Artificial Sequence) <220> <221> DOMAIN <223> Hu33VL2序列 <400> 43 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Asp Ile Asn Asn Phe 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 44 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu33 VL3 sequence <400> 44 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Asp Ile Asn Asn Phe 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Gly Ala Val Lys Leu Leu lie 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys 100 105 <210> 45 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu33 VH2 sequence <400> 45 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr lie Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gin Asp Asn Val 50 55 60 Gln Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gin Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 46 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu33 VH3 sequence <400> 46 Lys Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr lie Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gin Asp Asn Val 50 55 60 Gln Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gin Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 47 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23LCDR1 (N28Q) sequence <400> 47 Arg Ser Ser Gin Ser Leu Val His Ser Gin Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 48 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23LCDR1 (N28L) sequence <400> 48 Arg Ser Ser Gln Ser Leu Val His Ser Leu Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 49 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23LCDR1 (N28T) sequence <400> 49 Arg Ser Ser Gln Ser Leu Val His Ser Thr Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 50 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23LCDR1 (N28D) sequence <400> 50 Arg Ser Ser Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23LCDR1 (G29A) sequence <400> 51 Arg Ser Ser Gln Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu Glu 1 5 10 15 <210> 52 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 LCDR1 (G29V) sequence <400> 52 Asp lie Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 <210> 53 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL1 (N28Q) sequence <400> 53 Asp lie Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie65 70 75 80 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 54 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL1 (N28L) sequence <400> 54 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Leu Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 55 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL1 (N28T) sequence <400> 55 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 56 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23VL1 (N28D) sequence <400> 56 Asp Ile Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gin Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 57 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23VL1(G29A) sequence <400> 57 Asp Ile Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Asn Ala Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gin Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 58 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23VL1(G29V) sequence <400> 58 Asp Ile Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gin Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys 100 105 110 <210> 59 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL2 (N28Q) sequence <400> 59 Asp Gly Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gin Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys 100 105 110 <210> 60 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL2 (N28L) sequence <400> 60 Asp Gly Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Leu Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gin Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys 100 105 110 <210> 61 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL2 (N28T) sequence <400> 61 Asp Gly Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 62 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL2 (N28D) sequence <400> 62 Asp Gly Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 63 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Hu23 VL2 (G29A) sequence <400> 63 Asp Gly Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Ala Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 64 <211> 112 <212> PRT <213> 人工序列(Artificial Sequence) <220> <221> DOMAIN <223> Hu23VL2(G29V)序列 <400> 64 Asp Gly Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 65 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR1 consensus sequence for Hu32 and Hu23 antibodies <220> <221> DOMAIN <222> (4)..(4) <223> Xaa is selected from Ile or Met <400> 65 Asp Tyr Glu Xaa His 1 5 <210> 66 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR2 consensus sequence for Hu32 and Hu23 antibodies <220> <221> DOMAIN <222> (2)..(2) <223> Xaa is selected from Phe or Ile <220> <221> DOMAIN <222> (9)..(9) <223> Xaa is selected from Ile or Thr <220> <221> DOMAIN <222> (17)..(17) <223> Xaa is selected from Gly or Asp <400> 66 Leu Xaa Asp Pro Glu Thr Gly Gly Xaa Val Tyr Asn Gln Lys Phe Lys 1 5 10 15 Xaa <210> 67 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> General sequence for HCDR3 of Hu32 and Hu23 antibodies <220> <221> DOMAIN <222> (2)..(2) <223> Xaa is selected from Gly or Arg <220> <221> DOMAIN <222> (3)..(3) <223> Xaa is Phe or null <220> <221> DOMAIN <222> (4)..(4) <223> Xaa is Ser or null <220> <221> DOMAIN <222> (5)..(5) <223> Xaa is Tyr or null <220> <221> DOMAIN <222> (7)..(7) <223> Xaa is Gly or null <220> <221> DOMAIN <222> (8)..(8) <223> Xaa is Ser or null <220> <221> DOMAIN <222> (9)..(9) <223> Xaa is selected from Asn or Thr <220> <221> DOMAIN <222> (10)..(10) <223> Xaa is selected from Arg or Ser <400> 67 Glu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Trp Tyr Phe Asp Val 1 5 10 15 <210> 68 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR1 consensus sequence for Hu32 and Hu23 antibodies <220> <221> DOMAIN <222> (6)..(6) <223> Xaa is selected from He or Leu <220> <221> DOMAIN <222> (10)..(10) <223> Xaa is selected from Asn, Gin, Leu, Thr or Asp <220> <221> DOMAIN <222> (11)..(11) <223> Xaa is selected from Gly, Ala or Val <220> <221> DOMAIN <222> (12)..(12) <223> Xaa is selected from Asn or Lys <400> 68 Arg Ser Ser Gin Ser Xaa Val His Ser Xaa Xaa Xaa Thr Tyr Leu Glu 1 5 10 15 <210> 69 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR3 consensus sequence for Hu32 and Hu23 antibodies <220> <221> DOMAIN <222> (9)..(9) <223> Xaa is selected from Ala or Thr <400> 69 Phe Gin Gly Ser His Val Pro Tyr Xaa 1 5 <210> 70 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Heavy chain variable region consensus sequence for Hu32 and Hu23 antibodies <220> <221> DOMAIN <222> (26)..(26) <223> Xaa is selected from Gly or Asp <220> <221> DOMAIN <222> (27)..(27) <223> Xaa is selected from Gly, Phe or Tyr <220> <221> DOMAIN <222> (30)..(30) <223> Xaa is selected from Ser or Thr <220> <221> DOMAIN <222> (34)..(34) <223> Xaa is selected from He or Met <220> <221> DOMAIN <222> (38)..(38) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (43)..(43) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (48)..(48) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (51)..(51) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (58)..(58) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (66)..(66) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (67)..(67) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (68)..(68) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (70)..(70) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (83)..(83) <223> Xaa is selected from the group consisting of Arg, Lys, His, Gin, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Val, Leu, He, Phe, Tyr, Trp, Pro, Gly, and Ala <220> <221> DOMAIN <222> (97)..(97) <223> Xaa is selected from Ala or Thr <220> <221> DOMAIN <222> (100)..(100) <223> Xaa is selected from Gly or Arg <220> <221> DOMAIN <222> (101)..(101) <223> Xaa is Phe or null <220> <221> DOMAIN <222> (102)..(102) <223> Xaa is Ser or null <220> <221> DOMAIN <222> (103)..(103) <223> Xaa is Tyr or null <220> <221> DOMAIN <222> (105)..(105) <223> Xaa is Gly or null <220> <221> DOMAIN <222> (106)..(106) <223> Xaa is Ser or null <220> <221> DOMAIN <222> (107)..(107) <223> Xaa is selected from Asn or Thr <220> <221> DOMAIN <222> (108)..(108) <223> Xaa is selected from Arg or Ser <400> 70 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Xaa Xaa Thr Phe Xaa Asp Tyr 20 25 30 Glu Xaa His Trp Val Xaa Gin Ala Pro Gly Xaa Gly Leu Glu Trp Xaa 35 40 45 Gly Leu Xaa Asp Pro Glu Thr Gly Gly Xaa Val Tyr Asn Gin Lys Phe 50 55 60 Lys Xaa Xaa Xaa Thr Xaa Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Xaa Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Xaa Arg Glu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 71 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Light chain variable region consensus sequence for Hu32 and Hu23 antibodies <220> <221> DOMAIN <222> (2)..(2) <223> Xaa is selected from lie, Val or Gly <220> <221> DOMAIN <222> (29)..(29) <223> Xaa is selected from lie or Leu <220> <221> DOMAIN <222> (33)..(33) <223> Xaa is selected from Asn, Gin, Leu, Thr or Asp <220> <221> DOMAIN <222> (34)..(34) <223> Xaa is selected from Gly, Ala or Val <220> <221> DOMAIN <222> (35)..(35) <223> Xaa is selected from Asn or Lys <220> <221> DOMAIN <222> (102)..(102) <223> Xaa is selected from Ala or Thr <400> 71 Asp Xaa Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Xaa Val His Ser 20 25 30 Xaa Xaa Xaa Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Xaa Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 72 <211> 327 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> IgG4-AA heavy chain constant region sequence <400> 72 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 73 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Antibody Kappa Light Chain Constant Region Sequence <400> 73 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45 Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 74 <211> 452 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Hu23-11.IgG4 AA antibody heavy chain sequence <400> 74 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Leu lie Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gin Lys Phe 50 55 60 Lys Asp Arg Val Thr lie Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys 450 <210> 75 <211> 219 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Hu23-11.IgG4AA / Hu32a-85.IgG4AA / Hu23-11.IgG4P / Hu32a-85.IgG4P light chain sequence <400> 75 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 76 <211> 447 <212> PRT <213> 人工序列(Artificial Sequence) <220> <221> CHAIN <223> Hu32a-85.IgG4AA重链序列 <400> 76 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr 20 25 30 Glu Ile His Trp Val Lys Gln Ala Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 77 <211> 445 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Hu33-6.IgG4 AA heavy chain sequence <400> 77 Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gin Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 78 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Hu33-6.IgG4AA / Hu33-6.IgG4P light chain sequence <400> 78 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 79 <211> 327 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> IgG4-P heavy chain constant region sequence <400> 79 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser lie Gin Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg 210 215 220 Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys 225 230 235 240 Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 80 <211> 452 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Hu23-11. IgG4P antibody heavy chain sequence <400> 80 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys 450 <210> 81 <211> 447 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Hu32a-85. IgG4P heavy chain sequence <400> 81 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr 20 25 30 Glu Ile His Trp Val Lys Gln Ala Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 82 <211> 445 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Hu33-6. IgG4P heavy chain sequence <400> 82 Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gin Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445

Claims

1. A pharmaceutical composition comprising an anti-PD-1 antibody and a buffer, wherein: (a) the anti-PD-1 antibody has a concentration of 90 mg / mL to 150 mg / mL, the heavy chain variable region of the anti-PD-1 antibody comprises: HCDR1 as set forth in SEQ ID NO: 8, HCDR2 as set forth in SEQ ID NO: 9, and HCDR3 as set forth in SEQ ID NO: 10, and the light chain variable region comprises: LCDR1 as set forth in SEQ ID NO: 49, LCDR2 as set forth in SEQ ID NO: 12, and LCDR3 as set forth in SEQ ID NO: 13; (b) the buffer is an acetic acid-sodium acetate buffer or a histidine-acetate buffer, the buffer concentration is 5 mM to 30 mM, and the pH of the buffer is 4.7 to 5.7; (c) it further comprises an osmotic pressure regulator, wherein the osmotic pressure regulator is selected from sucrose, trehalose and sorbitol, and the concentration of the osmotic pressure regulator is 50 mg / mL to 100 mg / mL; (d) It further comprises a surfactant, wherein the surfactant is polysorbate 80, and the concentration of the polysorbate 80 is 0.6 mg / mL to 0.8 mg / mL.

2. The pharmaceutical composition according to claim 1, wherein the buffer is acetic acid-sodium acetate buffer.

3. The pharmaceutical composition according to claim 1, wherein the pH of the buffer is 5.

2. The pharmaceutical composition according to claim 1 , wherein the buffer concentration is 10 mM to 30 mM. The pharmaceutical composition according to claim 1 , wherein the buffer concentration is 10 mM. The pharmaceutical composition according to claim 1 , wherein the anti-PD-1 antibody concentration is 120 mg / mL or 100 mg / mL.

7. The pharmaceutical composition according to claim 1, wherein the concentration of polysorbate 80 is 0.6 mg / mL. The pharmaceutical composition according to claim 1 , wherein the osmotic pressure regulator is sucrose.

9. The pharmaceutical composition according to claim 1, wherein the concentration of the osmotic pressure regulator is 70 mg / mL to 90 mg / mL.

10. The pharmaceutical composition according to claim 1, wherein the concentration of the osmotic pressure regulator is 80 mg / mL. The pharmaceutical composition according to claim 1 , wherein the anti-PD-1 antibody has a heavy chain variable region as shown in SEQ ID NO: 27, and a light chain variable region as shown in SEQ ID NO:

55. The pharmaceutical composition according to claim 1 , wherein the anti-PD-1 antibody comprises a heavy chain constant region and a light chain constant region.

13. The pharmaceutical composition according to claim 12, wherein the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, and the light chain constant region is selected from human antibody kappa and lambda chain constant regions and conventional variants thereof. The pharmaceutical composition according to claim 13 , wherein the anti-PD-1 antibody comprises a heavy chain constant region as shown in SEQ ID NO: 72 and a light chain constant region as shown in SEQ ID NO:

73. The pharmaceutical composition according to claim 14 , wherein the anti-PD-1 antibody comprises: a heavy chain as shown in SEQ ID: 74, and a light chain as shown in SEQ ID:

75.

16. The pharmaceutical composition according to claim 1, comprising: A1) an anti-PD-1 antibody at a concentration of 90 mg / mL to 150 mg / mL, B1) acetic acid-sodium acetate buffer at a concentration of 10 mM to 30 mM and a pH of 4.7 to 5.7, C1) sucrose at a concentration of 70 mg / mL to 90 mg / mL, and D1) Polysorbate 80 at a concentration of 0.6 mg / mL to 0.8 mg / mL.

17. The pharmaceutical composition according to claim 1, comprising: 10 mM acetic acid-sodium acetate buffer with a pH of 5.2, an anti-PD-1 antibody at a concentration of 120 mg / mL, sucrose at a concentration of 80 mg / mL, and polysorbate 80 at a concentration of 0.6 mg / mL.

18. A pharmaceutical composition comprising: 10 mM acetic acid-sodium acetate buffer at a pH of 5.2, an anti-PD-1 antibody at a concentration of 120 mg / mL, sucrose at a concentration of 80 mg / mL, and polysorbate 80 at a concentration of 0.6 mg / mL; wherein, The anti-PD-1 antibody has a heavy chain as shown in SEQ ID: 74 and a light chain as shown in SEQ ID:

75.

19. A method for preparing the pharmaceutical composition according to any one of claims 1 to 18, comprising the step of subjecting the anti-PD-1 antibody stock solution to buffer replacement.

20. A lyophilized preparation containing an anti-PD-1 antibody, the lyophilized preparation being obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 18.

21. A reconstituted solution containing an anti-PD-1 antibody, wherein the reconstituted solution is obtained by reconstituted the lyophilized preparation according to claim 20.

22. A lyophilized preparation containing an anti-PD-1 antibody, wherein the lyophilized preparation can be reconstituted to form the pharmaceutical composition according to any one of claims 1 to 18.

23. A product comprising a container containing the pharmaceutical composition of any one of claims 1 to 18, the lyophilized formulation of claim 20 or 22, or the reconstituted solution of claim 21.

24. Use of the pharmaceutical composition of any one of claims 1 to 18, or the lyophilized formulation of claim 20 or 22, or the reconstituted solution of claim 21, or the preparation of claim 23 in the preparation of a medicament for treating a tumor, wherein the tumor is selected from the group consisting of head and neck cancer, central nervous system cancer, neuroendocrine tumors, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, hepatobiliary cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, and melanoma.

25. Use of the pharmaceutical composition of any one of claims 1 to 18, or the lyophilized formulation of claim 20 or 22, or the reconstituted solution of claim 21, or the preparation of claim 23 in the preparation of a medicament for treating a tumor, wherein the tumor is selected from the group consisting of head and neck squamous cell carcinoma, brain cancer, glioma, pharyngeal cancer, hepatoma, colorectal cancer, clear cell renal cell carcinoma, and Merkel cell carcinoma.

26. Use of the pharmaceutical composition of any one of claims 1 to 18, or the lyophilized formulation of claim 20 or 22, or the reconstituted solution of claim 21, or the preparation of claim 23 in the preparation of a medicament for treating a tumor, wherein the tumor is selected from liver cancer, gastric cancer, intestinal cancer, bone cancer, glioblastoma multiforme and nasopharyngeal carcinoma.

27. Use of the pharmaceutical composition according to any one of claims 1 to 18, or the lyophilized formulation according to claim 20 or 22, or the reconstituted solution according to claim 21, or the preparation according to claim 23 in the preparation of a medicament for treating a tumor, wherein the tumor is selected from colon cancer, chondrosarcoma and Ewing's sarcoma.

28. Use of the pharmaceutical composition of any one of claims 1 to 18, or the lyophilized formulation of claim 20 or 22, or the reconstituted solution of claim 21, or the product of claim 23 in the preparation of a medicament for treating a tumor, wherein the tumor is selected from: PD-L1-positive melanoma, lung cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, and intestinal cancer.

29. Use of the pharmaceutical composition of any one of claims 1 to 18, or the lyophilized formulation of claim 20 or 22, or the reconstituted solution of claim 21, or the preparation of claim 23 in the preparation of a medicament for treating a tumor, wherein the tumor is selected from: PD-L1-positive non-small cell lung cancer and colon cancer.

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