KR20220001106A - High concentration anti-VEGF antibody formulation and anti-VEGF antibody for use in the same - Google Patents

Abstract

Disclosed are a stable aqueous pharmaceutical composition containing high concentrations of anti-VEGF antibody and anti-VEGF antibodies suitable for use in such composition. More specifically, the present invention relates to a stable aqueous pharmaceutical composition which comprises a humanized antibody against a vascular endothelial growth factor (VEGF) as an active ingredient at a concentration of 150 mg/ml or more.

Description

High concentration anti-VEGF antibody formulation and anti-VEGF antibody for use in the same

The present disclosure relates to a high concentration antibody formulation, and more particularly, to a stable aqueous pharmaceutical composition containing a high concentration of an anti-VEGF antibody and an anti-VEGF antibody suitable for use in the composition.

Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that promote blood vessel formation, and is involved in vasculogenesis and angiogenesis. The normal function of VEGF is to create new blood vessels during embryogenesis or to bypass clogged blood vessels after trauma or after trauma, but it also contributes to disease. Cancer cells express VEGF to cause growth and metastasis, and overexpression of VEGF can cause vascular disease in the retina of the eye. VEGF is secreted from retinal epithelial cells in hypoxia and increases the differentiation of vascular endothelial cells and the permeability of retinal blood vessels. It is known to play an important role in the increase. Therefore, blocking vascular endothelial growth factor is an important treatment in age-related macular degeneration, and several vascular endothelial growth factor antagonists are being used. Representative examples include bevacizumab (Avastin®), ranibizumab (Ranibizumab, Lucentis®), and aflibercept (Aflibercept, Eylea®).

The concentration of therapeutic antibody in liquid pharmaceutical formulations varies greatly, for example, depending on the route of administration. High concentration formulations may be desirable for intravitreal injection. However, formulations with high antibody concentrations may have a short shelf life, and formulated antibodies may lose biological activity due to chemical and physical instability during storage. Aggregation, deamidation and oxidation are known to be the most common causes of antibody degradation. In particular, aggregation should be minimized or prevented, as it can potentially increase the patient's immune response and raise safety concerns.

Methods for preparing high concentration VEGF antagonist formulations are disclosed. International Patent Publication No. WO2013/063510 discloses an antibody, including bevacizumab, ranibizumab, and the like, as an amino acid for stabilizing the antibody, valine; additional amino acids selected from the group consisting of serine, threonine, alanine and glycine; and additional amino acids selected from the group consisting of isoleucine, asparagine, glutamine and aspartic acid; and proline as an amino acid for reducing the viscosity of the formulation, a stable pharmaceutical formulation comprising an amino acid, a viscosity of less than 100 cP, and free of arginine and histidine. Korean Patent Application Laid-Open No. 2020-0029374 discloses a liquid composition comprising aflibercept, a stabilizer including at least one selected from the group consisting of polyols and sugars, and a surfactant, and having a pH of 4 to 8. Korea Patent Publication No. 2017-0076781 discloses an aqueous solution comprising at least 50 mg/ml of an anti-VEGF antibody, Brolucizumab, sucrose or trehalose, citrate or histidine buffer, and polysorbate 80 as a surfactant. A pharmaceutical composition is disclosed.

Despite the disclosure of the above documents, there has been a need for a stable aqueous pharmaceutical composition containing a high concentration of anti-VEGF antibody and having a low viscosity.

International Patent Publication No. WO2013/063510 Korean Patent Publication No. 2020-0029374 Korean Patent Publication No. 2017-0076781

It is an object of the present disclosure to provide a stable aqueous pharmaceutical composition containing an anti-VEGF antibody in a high concentration.

Another object of the present disclosure is to provide an anti-VEGF antibody suitable for use in the composition.

One aspect of the present disclosure provides a stable aqueous pharmaceutical composition comprising a humanized antibody to vascular endothelial growth factor (VEGF) as an active ingredient at a concentration of 150 mg/ml or more.

Another aspect of the present disclosure provides an anti-VEGF antibody suitable for use in the composition, one or more of the following antibodies:

i) a heavy chain variable region having a heavy chain framework consisting of HFR1 of SEQ ID NO: 39, HFR2 of SEQ ID NO: 40, HFR3 of SEQ ID NO: 41, and HFR4 of SEQ ID NO: 42; And comprising a light chain variable region having a light chain framework consisting of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 44, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46, a humanized anti-VEGF antibody;

ii) a heavy chain variable region having a heavy chain framework consisting of HFR1 of SEQ ID NO: 53, HFR2 of SEQ ID NO: 54, HFR3 of SEQ ID NO: 55, and HFR4 of SEQ ID NO: 42; And comprising a light chain variable region having a light chain framework consisting of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 56, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46, a humanized anti-VEGF antibody; and,

iii) a heavy chain variable region of SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37; And comprising the light chain variable region of SEQ ID NO: 34, a humanized anti-VEGF antibody.

According to the present disclosure, a stable aqueous pharmaceutical composition having a low viscosity while containing a high concentration of the anti-VEGF antibody and an anti-VEGF antibody suitable for use in the composition are provided.

1 is a diagram comparing the amino acid sequences of rabbit and humanized anti-VEGF antibody.
2 is a diagram comparing the amino acid sequences of ranibizumab and mutated ranibizumab.
3 is a graph showing the VEGF binding ability of the anti-VEGF antibody.
4 is a graph showing the calcium inhibitory effect induced by VEGF in HUVEC cells of the anti-VEGF antibody.
5 is a view showing the results of analysis of the aggregate content of anti-VEGF antibody.
6 is a graph showing the results of measuring the viscosity at a high concentration of the anti-VEGF antibody.
7 is a diagram showing the binding ability of the anti-VEGF antibody to FcγRI, II, III.

According to one aspect of the present disclosure, there is provided a stable aqueous pharmaceutical composition comprising a humanized antibody against vascular endothelial growth factor (VEGF) at a high concentration as an active ingredient.

As used herein, the term “aqueous pharmaceutical composition” refers to a composition suitable for pharmaceutical use containing an aqueous carrier. Compositions suitable for pharmaceutical use may be sterile, homogeneous and/or isotonic. Aqueous pharmaceutical composition means, for example, a liquid formulation or a lyophilized formulation to be reconstituted.

The term “stable” pharmaceutical composition refers to an agent that preserves stability, including physical stability. A variety of analytical techniques are available in the art, such as visual inspection, SDS-PAGE, IEF, size exclusion chromatography, reversed phase liquid chromatography, ion exchange chromatography, capillary electrophoresis, light scattering, particle counting, turbidity, etc. It can be determined by the above method. In an embodiment, stability is assessed for aggregate formation. For example, a stable aqueous pharmaceutical composition is less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1% as measured by gel filtration chromatography. It may be to form the following aggregates.

In an embodiment, the concentration of anti-VEGF antibody in the aqueous pharmaceutical composition may be at least 150 mg/ml, for example between 150 and 300 mg/ml. The aqueous pharmaceutical composition can be, for example, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 210 mg/ml, about 220 mg/ml, about 230 mg/ml, about 240 mg/ml, about 250 mg/ml, about 260 mg/ml, about 270 mg/ml, about 280 mg/ml, about 290 mg/ml or about 300 mg/ml of anti-VEGF antibody may be included.

As used herein, the term “antibody” includes a whole antibody and any antigen-binding fragment or single chain thereof. "Antibody" includes a glycoprotein comprising at least two heavy (H) chains and two light (L) chains or antigen-binding portions thereof interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH or HV) and a heavy chain constant region. The heavy chain constant region consists of three regions, namely CH1, CH2 and CH3. Each light chain consists of a light chain variable region (VL or LV) and a light chain constant region. The light chain constant region consists of one region, namely CL. The VH and VL regions can be subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FR). each of VH and VL is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (for VH, HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4; for VL, LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4), consisting of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions of the heavy and light chains contain binding regions that interact with antigens. The constant regions can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q).

As used herein, the term "humanized antibody" refers to a molecule wherein the antigen-binding portion of the molecule is substantially derived from an immunoglobulin from a non-human species, while the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. points to The antigen-binding portion may comprise a complete variable region fused onto a constant region or may comprise only a complementarity determining region (CDR) grafted to an appropriate framework region within the variable region. The antigen-binding portion may be wild-type or modified by one or more amino acid substitutions, eg, modified to more closely resemble a human immunoglobulin. Some types of humanized antibodies preserve all CDR sequences (eg, a humanized rabbit antibody containing all six CDRs from a rabbit antibody). Other types may differ in one or more CDRs from the original antibody.

In an embodiment, the antibody comprises a heavy chain variable region having a heavy chain framework having one or more of Kabat numbering 13Q, 37I, 74S, 77T and 105Q as compared to human germ-line immunoglobulin; a light chain variable region having a light chain framework having at least one of 22T, 43A, 83F and 100Q; Or it may include a combination thereof.

Additionally, the heavy chain framework may be one having one or more of 49A and 73D.

For example, the antibody has a heavy chain framework of one or more of HFR1 of SEQ ID NO:39 or SEQ ID NO:53, HFR2 of SEQ ID NO:40 or SEQ ID NO:54, HFR3 of SEQ ID NO:41 or SEQ ID NO:55, and HFR4 of SEQ ID NO:42 heavy chain variable region; a light chain variable region having a light chain framework of at least one of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 44 or SEQ ID NO: 56, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46; Or it may include a combination thereof.

For example, the antibody comprises a heavy chain variable region having one or more heavy chain CDRs of HCDR1 of SEQ ID NO: 47 or SEQ ID NO: 57, HCDR2 of SEQ ID NO: 48 or SEQ ID NO: 58, and HCDR3 of SEQ ID NO: 49 or SEQ ID NO: 59; a light chain variable region having at least one light chain CDR of LCDR1 of SEQ ID NO: 50 or SEQ ID NO: 60, LCDR2 of SEQ ID NO: 51 or SEQ ID NO: 61, and LCDR3 of SEQ ID NO: 52 or SEQ ID NO: 62; Or it may include a combination thereof.

For example, the antibody comprises a heavy chain variable region of SEQ ID NO: 30; And it may include a light chain variable region of SEQ ID NO: 31.

For example, the antibody comprises a heavy chain variable region of SEQ ID NO: 33; and a light chain variable region of SEQ ID NO: 34.

In embodiments, the antibody may have reduced or eliminated effector function. For example, the antibody may have reduced or eliminated binding ability to one or more of FcγRI, FcγR II, FcγR III, and FcRn. For example, the antibody may have one or more mutations among L234A, L235A, P331G, 1253A, and H310A. For example, the antibody may be one comprising the heavy chain of SEQ ID NO: 32 or SEQ ID NO: 38.

In an embodiment, the antibody may have _{a binding affinity for VEGF of K D} 100 pM or less. For example, the antibody has a K _D for VEGF of 50 pM or less, 30 pM or less, 20 pM or less, 15 pM or less, 10 pM or less, 5 pM or less, 1-50 pM, 1-30 pM, 1-20 pM, It may have a binding affinity of 1 to 15 pM, 1 to 10 pM, or 1 to 5 pM.

In an embodiment, the antibody may have a thermal stability of at least 70 °C Tm. For example, an antibody may have a Tm of 71 °C or higher, Tm 72 °C or higher, Tm 73 °C or higher, Tm 74 °C or higher, Tm 75 °C or higher, Tm 76 °C or higher, Tm 77 °C or higher, Tm 70-80 °C or higher, Tm 75- It may have a thermal stability of 80 ℃, Tm 75 ~ 77 ℃.

In an embodiment, the aqueous pharmaceutical composition may include, in addition to the active ingredient, a buffer and a tonicity agent.

A buffer is, for example, maintaining a pH in the range of 5.0 to 7.5, for example, the aqueous pharmaceutical composition may have a pH of 5.0 to 7.5, 5.0 to 7.0, 5.0 to 6.5, 5.0 to 6.0, 5.0 to 5.5. Examples of buffers are one or more of the conventional ones known in the art, and include salts of organic acids such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; tris, tromethamine hydrogloide or phosphate buffers, amino acid buffers such as histidine. In certain instances, the buffer may be a histidine buffer. For example, the buffer may have a concentration in the range of 1-50 mM, 5-50 mM, 5-40 mM, 5-30 mM, 5-20 mM, 5-15 mM.

For example, the isotonic agent may be a salt. The salt may include at least one selected from the group consisting of NaCl, KCl, NaF, KBr, NaBr, Na ₂ SO ₄ , NaSCN, and K ₂ SO _{4 , and in particular may be NaCl.} For example, the salt may have a concentration in the range of 50-200 mM, 75-175 mM, 100-150 mM. In certain instances, the isotonic agent may be a sugar or sugar alcohol, or not a polyol. Examples of sugars or sugar alcohols, or polyols, may include trehalose, sucrose, mannitol, sorbitol, xylitol, glucose, glycerol, and the like.

In embodiments, the aqueous pharmaceutical composition may be free of stabilizers, surfactants, or both. The surfactant may be a nonionic surfactant, for example, polysorbates (eg, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene ( 20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate); The numerical value (20) of oxyethylene groups ( _{meaning the total number of oxyethylene groups (-(CH 2} CH ₂ O)-)), poloxamer (PEO-PPO-PEO copolymer; PEO: poly(ethylene oxide), PPO: poly (propylene oxide)), polyethylene-polypropylene glycol, polyoxyethylene compounds (eg, polyoxyethylene-stearate, polyoxyethylene alkyl ether (alkyl: C ₁ -C ₃₀ ), polyoxyethylene monolauryl ether, alkyl It may be at least one selected from the group consisting of phenyl polyoxyethylene copolymer (alkyl: C ₁ -C ₃₀ ), etc.), sodium dodecyl sulphate (SDS), and the like. For example, the surfactant may be polysorbates (eg, polysorbate 20). The stabilizer may include one or more amino acids or a salt thereof, for example, an amino acid other than histidine or a salt thereof. Examples of the amino acid may include valine, serine, threonine, alanine, glycine, isoleucine, asparagine, glutamine, aspartic acid, and proline. In certain instances, the aqueous pharmaceutical composition may be proline-free. In another specific example, the aqueous pharmaceutical composition may comprise proline.

In certain embodiments, the aqueous pharmaceutical composition comprises or consists essentially of 5-50 mM histidine buffer and 50-200 mM NaCl with an anti-VEGF antibody. (consisting of) may be.

In an embodiment, the aqueous pharmaceutical composition may have a viscosity of 20 cP or less at 25 °C. The viscosity may be within the range of 1 to 20 cP, 2 to 18 cP, 2 to 15 cP, 3 to 15 cP, 2 to 10 cP, 3 to 10 cP, 5 to 15 cP, 5 to 10 cP at 25 ° C. .

In an embodiment, an aqueous pharmaceutical composition comprising an anti-VEGF antibody may be used to treat a variety of diseases or disorders. A pharmaceutical composition comprising an anti-VEGF antibody is useful for treating an angiogenic disease, particularly an angiogenic ocular disease, in a subject or patient. "Angiogenic eye diseases" treatable using the aqueous pharmaceutical composition according to the present disclosure include abnormal angiogenesis, choroidal neovascularization (CNV), retinal vascular permeability, retinal edema, diabetic retinopathy (particularly proliferative diabetic retinopathy). ), diabetic macular edema, neovascular (exudative) age-related macular degeneration (AMD) including CNV associated with nAMD (neovascular age-related macular degeneration (AMD)), sequelae associated with retinal ischemia, central retinal vein occlusion (CRVO) and conditions, diseases or disorders associated with ocular neovascularization, including, but not limited to, posterior segment neovascularization.

In an embodiment, the aqueous pharmaceutical composition may comprise additional active ingredients in addition to the anti-VEGF antibody. Additional pharmacological agents may include, for example, other antibodies useful for the treatment of ocular diseases.

In an embodiment, the aqueous pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration (eg, injection), intraocular administration (eg, intravitreal administration), intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration Administration, or intratumoral administration, etc. may be used, and in particular, it may be administered by intravitreal administration. In a specific example, the aqueous pharmaceutical composition may be an ophthalmic composition comprising an anti-VEGF antibody, and in this case, may be an injection administered into the vitreous of the eye.

In an embodiment, the pharmaceutical composition according to the present disclosure may be administered at an appropriate administration cycle and/or dose according to the judgment of an expert such as an ophthalmologist according to the patient's condition, including age, health status, disease severity, and the like. Since the composition may contain a high concentration of the anti-VEGF antibody, the amount of the active ingredient administered at one time may be increased, and thus may be administered less frequently, that is, at a longer cycle. For example, the composition may be administered at an administration cycle of once every 3 months or more, once every 4 months, once every 5 months, or once every 6 months or more. In addition, the composition can increase the amount of the active ingredient administered per volume, for example, it can be administered in a dose ranging from 2 to 30 mg. For example, it may be administered in a dose of about 2 mg, 5 mg, 7.5 mg, 10 mg, 15 mg, 20 mg, 25 mg, or 30 mg. The dose may be administered in various volumes suitable for intraocular administration, such as 100 μl or less, 50-100 μl, 50 μl or less.

In an embodiment, the aqueous pharmaceutical composition may be in the form of a solution or a lyophilized powder. The composition may be contained in a vial, an ampoule, or a pre-filled syringe. In certain instances, lyophilization is contemplated to provide an aqueous pharmaceutical composition of the present disclosure. Lyophilization techniques are known in the art. The lyophilisate must be reconstituted with an aqueous reconstitution agent prior to administration to the patient. This step makes it possible to redissolve the antibody and other components in the lyophilisate to provide a solution suitable for injection into the patient. The volume of aqueous medium used for reconstitution determines the concentration of antibody in the resulting pharmaceutical composition. Reconstitution with a volume of reconstitution agent smaller than the volume prior to lyophilization provides a more concentrated composition than prior to lyophilization. The reconstitution factor (volume of formulation after lyophilization: volume of formulation before lyophilization) may be 1:0.5 to 1:6. As described above, the lyophilisate of the present invention can be reconstituted to provide an aqueous composition having an anti-VEGF antibody concentration of at least 150 mg/ml, for example 150-300 mg/ml, wherein the volume of the reconstituted agent is thus will be selected accordingly. If necessary, the reconstituted formulation can be diluted prior to administration to a patient when appropriate to deliver the intended dosage. Conventional reconstitution agents include sterile water or buffers, optionally containing a preservative. Where the lyophilisate comprises a buffer, the reconstitution agent may comprise an additional buffer (which may be the same or different from the buffer of the lyophilisate) or a buffer (e.g., WFI (water for injection) or physiological saline). may not

Examples of other excipients that may be included in the aqueous pharmaceutical composition of the present disclosure include antibacterial agents, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin. , gelatin, casein, salt forming counterions such as sodium and the like.

Another aspect of the present disclosure provides a humanized anti-VEGF antibody suitable for use in an aqueous pharmaceutical composition containing a high concentration of anti-VEGF.

The antibody comprises a heavy chain variable region having a heavy chain framework consisting of HFR1 of SEQ ID NO: 39, HFR2 of SEQ ID NO: 40, HFR3 of SEQ ID NO: 41, and HFR4 of SEQ ID NO: 42; And it may include a light chain variable region having a light chain framework consisting of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 44, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46.

In an embodiment, the antibody comprises a heavy chain variable region of SEQ ID NO: 30; And it may include a light chain variable region of SEQ ID NO: 31.

In an embodiment, the antibody may include the heavy chain of SEQ ID NO: 32.

In an embodiment, the antibody comprises a heavy chain variable region having a heavy chain framework consisting of HFR1 of SEQ ID NO: 53, HFR2 of SEQ ID NO: 54, HFR3 of SEQ ID NO: 55, and HFR4 of SEQ ID NO: 42; And it may include a light chain variable region having a light chain framework consisting of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 56, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46.

In an embodiment, the antibody comprises a heavy chain variable region of SEQ ID NO: 33; and a light chain variable region of SEQ ID NO: 34.

In an embodiment, the antibody may include the heavy chain of SEQ ID NO: 38.

In an embodiment, the antibody comprises a heavy chain variable region of SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37; and a light chain variable region of SEQ ID NO: 34.

Another aspect of the present disclosure is a disease or disorder mediated by VEGF comprising administering to a subject or patient in a therapeutically effective amount of the aqueous pharmaceutical composition or the humanized anti-VEGF antibody, e.g. Methods are provided for treating eg angiogenic diseases, eg, neovascular ocular diseases, in particular age related macular degeneration.

The term "therapeutically effective amount" means an amount sufficient to produce a therapeutic effect when administered to a subject or patient in need of treatment.

The term "treating or treatment" means treating a disease or medical condition in a subject, such as a mammal, including a human, including:

(a) inhibiting the disease or medical condition, ie, slowing or arresting the progression of the disease or medical condition in a subject; or

(b) alleviating the disease or medical condition in the subject.

As used herein, the term "subject" or "patient" refers to human and non-human mammals including, but not limited to, primates, rabbits, pigs, horses, dogs, cats, sheep and cattle. For example, the subject or patient is a human.

A further aspect of the present disclosure provides a method of delivering an anti-VEGF antibody to a subject or human, comprising administering the aqueous pharmaceutical composition or the humanized anti-VEGF antibody to the subject or patient.

A further further aspect relates to said aqueous pharmaceutical composition or said humanized antibiotic for the manufacture of a medicament for the treatment of a disease or disorder mediated by VEGF, for example an angiogenic disease, for example an angiogenic eye disease, in particular age-related macular degeneration. Provided is a use of the -VEGF antibody.

Still another aspect relates to the use of said aqueous pharmaceutical composition or said humanized anti-VEGF antibody to treat a disease or disorder mediated by VEGF, eg an angiogenic disease, eg an angiogenic eye disease, in particular age-related macular degeneration. It provides a method for preparing a medicament for

Hereinafter, the present invention will be described in detail with reference to Examples, but this is only intended to help the understanding of the present invention and is not intended to limit the scope of the present invention in any way.

<Example 1> Rabbit Immunization and Immune Library Preparation

Four rabbits (New Zealand White) were immunized with VEGF by intraperitoneal administration of VEGF165 (Sino biological, CA) 4 times. RNA was isolated by receiving spleen and bone marrow extracts from 4 immunized rabbits. cDNA was synthesized using the GoScripts kit (Promega, USA) according to the manufacturer's instructions. The mRNA used at this time was 2 μg.

Using the cDNA as a template, the variable region (VL and VH) genes of the rabbit antibody were amplified. First, in order to amplify the light chain variable region (VL) of a rabbit antibody, 5'-specific primers and SEQ ID NOs: 4 and 5 of the rabbit light chain variable region as SEQ ID NOs: 1, 2, 3 and 7 using the cDNA as a template , 6, and 8, respectively, using 3'-specific primers in pairs, PCR was performed to selectively amplify the rabbit antibody light chain gene. In addition, in order to amplify the heavy chain variable region (VH) of the rabbit antibody, 5' specific primers and SEQ ID NOs: 13 and 14 of the rabbit heavy chain variable region of SEQ ID NOs: 9, 10, 11, and 12 using the cDNA as a template PCR was performed to selectively amplify the rabbit antibody heavy chain gene by using the 3'-specific primers described as a pair, respectively. Finally, in order to amplify the scFv linker part, the 5' specific primers set forth in SEQ ID NOs: 15, 16, and 17 and the 3' specific primers set forth in SEQ ID NOs: 18, 19, 20, and 21 using pCom3XSS-scFv DNA as a template PCR was performed using each pair of red primers to selectively amplify the linker gene.

Extension PCR was performed using each VL, linker, and VH gene amplified in the PCR as a template. First, for the extension PCR of the VL gene, 100 ng of the VL, linker, and VH genes amplified in the PCR, the primers shown in SEQ ID NO: 22 and the primers shown in SEQ ID NO: 23 were used. These PCR reaction conditions were repeated 30 times using Ex-Taq DNA polymerase (Takara Co., Ltd.) after pre-denaturing at 95°C for 5 minutes, at 95°C for 50 seconds, at 55°C for 50 seconds, and at 72°C for 1 minute. .

The VL-Linker-VH (scFv) gene and the pCom3XSS vector obtained above were each digested with a restriction enzyme SfiI, purified, and the two cut DNA fragments were ligated and then the enzyme was inactivated. After eluting with distilled water using a XXPC DNA concentration column, the library DNA was transformed into E. coli ER2738 by electroporation.

After culturing in SOC medium, put it in a 2 L flask with 500 ml SB + carbenicillin (50 μg/ml) and M13K07 helper phage (MOI: 1:20), let stand at 37° C. for 30 minutes, and add kanamycin to 50 μg/ml mL, and incubated with shaking at 30 °C overnight. The next day, the culture medium was centrifuged to precipitate cells, and only the library phage in the supernatant was collected.

<Example 2> Phage library screening

Panning and screening were performed using the rabbit immune library phage prepared above. Bio-panning was performed three times with an Immunotube (Immuno™tube) coated with 5 μg/ml recombinant human VEGF fusion protein to select clones reactive to human VEGF. 96 phage clones were randomly selected from the colonies grown on the output plate, and their reactivity to human VEGF was tested through a phage enzyme immunoassay.

DNA was sequenced for the finally selected clones and classified into 6 scFv clones identified as having different complementarity determinant sequences. Afterwards, one final type of 2-19 clone was selected through an enzyme immunoassay to confirm VEGF binding ability, which is described in SEQ ID NO: 26 and SEQ ID NO: 27, which encode the amino acid sequence shown in SEQ ID NO: 24 and SEQ ID NO: 25 It was confirmed that the nucleotide sequence was encoded.

<Example 3> Preparation of humanized antibody against rabbit anti-VEGF antibody

For humanized antibody design, human germ-line sequences most similar to the heavy chain variable region and light chain variable region of clones 2-19 were identified in IMGT/V-QUEST. In the case of the heavy chain variable region, it was confirmed that IGHV3-21 and IGHV3-53, and in the case of the light chain, IGKV1-27 showed the highest similarity. Based on this sequence, most of the framework parts were replaced with human sequences and heavy chain sequences capable of affecting antigen binding, rabbit sequences for Kabat Nos. D28, D30, V or A for V24, V or I for V37, In the case of G49, the rabbit sequence and human sequence were entered as G or A, in the case of S73, S or D, and in the case of F91, the human sequence and the rabbit sequence were inserted. The light chain was designed to contain rabbit and human sequences as N or T for Kabat number N22, P or A for P43, L or I for L48, V or F for A83 and F or Y for F91 . Figure 1 shows the comparison of amino acids of rabbit and humanized anti-VEGF antibody. In FIG. 1, boldface indicates the human sequence, and the underlined portion indicates an amino acid contributing to solubility enhancement.

In order to convert the heavy and light chain parts and 2-19 scFv forms to be converted into humanized sequences into full IgG form, PCR (assembly PCR) was performed in which synthetic oligo primers were combined using SEQ ID NO: 26 and SEQ ID NO: 27 as templates. According to the instructions in TAKARA's Infusion kit for the animal expression vector pCEP6-WPRE (the vector in which the Fc region is inserted), the humanized antibody light chain expression vector and heavy chain expression vector 2-19 were prepared in the same way. It was confirmed through DNA sequencing. SEQ ID NOs are as follows: Hz1 HV; SEQ ID NO: 28, Hz1 LV; SEQ ID NO: 29, Hz2 HV; SEQ ID NO: 30, Hz2 LV; SEQ ID NO: 31.

<Example 4> Preparation of mutant anti-VEGF antibody for enhancing the activity and hydrophilicity of ranibizumab

A phage library was prepared in order to replace the framework partial sequence of the heavy chain variable region of the Hz2 antibody in order to convert from the ranibizumab (Lucentis) Fab form to the whole antibody form, increase activity and improve hydrophilicity. For the framework portion of the heavy chain variable region, based on the Hz2 sequence, Kabat Nos. A24, V27, G49, F69, L71, T73, S76, A78, K94 sequences V, I, A, F, L, T, S, A, A primer was prepared so as to be substituted with R, and library preparation and screening were performed by the method of Example 2. As a result, 10 candidate groups were secured, among which 4 clones with good binding affinity, Lu007 (SEQ ID NO: 33, SEQ ID NO: 34), F8 (SEQ ID NO: 35, SEQ ID NO: 34), E9 (SEQ ID NO: 36, SEQ ID NO: 34) , H2 (SEQ ID NO: 37, SEQ ID NO: 34) was selected. In FIG. 2, amino acid sequences of Lucentis and mutated Lucentis were compared. In FIG. 2, boldface indicates the Hz2 sequence, and the underlined portion indicates Hz2 amino acid and HCDR3 modified amino acid.

<Example 5> Antibody expression and purification

Humanized antibody expression was performed using the ExpiCHO-S system (Thermo). The general culture of hamster uterine cells ExpiCHO-S was performed according to the manufacturer's instructions. After 12 days, the culture medium was recovered, centrifuged at 5000 rpm, 15 minutes, and room temperature, passed through a 0.22 μm Amicon Ultra-15 (Millipore) filter to remove cell debris, and then AKTA pure device and Mabselectsure (GE) column was used for purification.

<Example 6> ELISA to confirm VEGF binding ability

ELISA was performed to confirm the binding ability to VEGF. First, 100 μl of hVEGF was added to an immunoplate at a concentration of 500 ng/ml and reacted at room temperature for 1 hour. After preparing by dissolving 2% skim milk in PBST, 200 μl of VEGF-coated wells were added and reacted at room temperature for 2 hours. After the reaction, well 3 was washed with 300 μl of PBST, and Hz1 (humanized antibody prepared as SEQ ID NO: 28, SEQ ID NO: 29), Hz2 (humanized antibody prepared as SEQ ID NO: 30, SEQ ID NO: 31) purified above, RIgG1 (rabbit antibody prepared as SEQ ID NO: 24, SEQ ID NO: 25), Lu007 (SEQ ID NO: 33, SEQ ID NO: 34), F8 (SEQ ID NO: 35, SEQ ID NO: 34), E9 (SEQ ID NO: 36, SEQ ID NO: 34), H2 (SEQ ID NO: 37, SEQ ID NO: 34) Started at a concentration of 100 ng/ml and diluted two-fold to make a total of 11 samples, put into VEGF-coated wells, and reacted at room temperature for 20 minutes. After the reaction, wells were washed 3 times with 300 μl of PBST, and then anti-hIgG-Fc HRP (1:3000, Thermo) was added at 100 μl/well, reacted for 30 minutes, and washed 3 times as above, followed by TMB (BioFx) solution was added at 100 μl/well, and the color was developed at room temperature for 5 minutes. After color development, 50 μl/well of 2 M sulfuric acid was added to stop color development, and absorbance was measured at 450 nm. The results are shown in FIG. 3 .

As shown in FIG. 3 , it was confirmed that all of Hz1, Hz2 and RigG1, a rabbit antibody, bind to VEGF without any difference in VEGF binding ability. In addition, as shown in FIG. 3 , it was confirmed that the variant showed better binding ability than that of Lucentis-Ig or Lu007.

<Example 7> Determination of antibody affinity using octets

Affinity determination for VEGF was performed as follows. Octet red96E was used, and human VEGF immobilized on the biosensor AR2G and RigG1, Hz1, Hz2, Lu007, and Lucentis-Ig used as analytes interacted and the binding and dissociation values were compared and measured. For human VEGF, recombinant human VEGF165 (Sino biological, CA) was used, and VEGF was immobilized on the AR2G sensor chip by amine coupling. At this time, the amount of VEGF used was 10 μg/ml, and the degree of coating was confirmed with a wavelength of 5 nm. In order to check the reactivity according to each concentration, the antibody concentrations were measured as 25, 12.5, 6.25, 3.12, 1.56, 0.78 nM. During the measurement, the rotation speed was set to 100, and the temperature was measured at 30 °C. The concentration of the sample was adjusted using a kinetic buffer (Molecular Device). The antibody solution of each concentration was used as a binding phase for 5 minutes, and then switched to a kinetic buffer to be a dissociated phase for 30 minutes. Measurements for each antibody are shown in Table 1. As a result, it was confirmed that RigG1, Hz1 and Hz2 all showed very good affinity of 5 pM or less, and Lu007 and Lucentis-Ig also showed excellent affinity at 12 and 14 pM levels.

Affinity measured using octets Replicate Group K _D (M) kon(1/Ms) kdis(1/s) RigG1 3.98E-12 7.75E+05 3.08E-06 Hz1 4.71E-12 6.85E+05 3.22E-06 Hz2 1.93E-12 5.05E+05 9.73E-07 Hz2-F 3.98E-12 7.75E+05 3.08E-06 Lu007 1.22E-11 2.37E+05 2.89E-06 Lucentis-Ig 1.48E-11 4.00E+05 5.92E-06

<Example 8> Comparison of Calcium Inhibition Efficacy Induced by VEGF in HUVEC Cells of Anti-VEGF Antibodies

In order to compare the VEGF inhibitory efficacy of the antibody, the amount of calcium increase by VEGF in HUVEC cells was compared. Comparative tests were performed using Hz1, Hz2, aflibercept (Eylea), bevacizumab (Avastin), Lucentis-Ig, and Lu007 samples. HUVEC (LONZA, C2519AS) cells maintained at passage 2-7 in EBM-2 (Lonza-Clonetics, Walkersville, MD) containing EGM-2 SingleQuots (Lonza-Clonetics, Walkersville, MD, #CC-4176) were used. , HUVEC cells were seeded at 30,000 cells/well on a black 96-well plate (NUNC, 165305) coated with 0.1% collagen and cultured for one day, followed by calcium analysis. No purified material is added, or 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 or 20 nM is mixed with VEGF165 (R&D systems, 293-VE-500) 1.5 nM and then incubated at room temperature for 30 minutes. Antigen binding was induced. To measure the amount of intracellular calcium, a calcium assay kit (BD, #640176) was used according to the manufacturer's instructions, and a calcium indicator dye was added to each well and incubated at 37°C for 1 hour. Mixture treatment and changing the amount of calcium in HUVEC cells were measured using Flexstation Ⅲ ROM v2.1.28, Molecular Devices, and 50 μl of the treated material was added to the cell plate by an automatic mechanism and then the calcium indicator dye was removed from the cell plate. The resulting fluorescence (excitation=485 nm; emission=525 nm) was recorded every 3 s over a period of 150 s. The results are shown in FIG. 4 . As shown in FIG. 4, it was confirmed that Hz1, Hz2, Lu007, and Lucentis-Ig had an equivalent IC50 value in the range of 0.8-0.87 nM, which is almost similar to commercially available aflibercept, which is 1.2 nM avastin. it was better than

<Example 9> Viscosity measurement and aggregate measurement at high concentration of Hz1 and Hz2

The most problematic thing when concentrating protein at a high concentration is a sudden increase in viscosity. In this respect, the importance of viscosity after concentration can be confirmed. It is generally known that the acceptable viscosity range for protein preparations is 20 cP or less. Viscosity measurement and aggregate formation after high concentration concentration were performed as follows.

The purified Hz1 and Hz2 were concentrated by an ultrafiltration membrane in a solution of 10 mM sodium phosphate, 150 mM NaCl, pH 6.2, and concentrated to a final concentration of 200 mg/ml. Comparing the time of the enrichment process, it was confirmed that Hz1 requires a longer period of enrichment than Hz2. After the completion of concentration, the presence or absence of the formation of aggregates was first confirmed by gel filtration chromatography. The conditions are as follows. A SEC-3 300A, 7.8X300 mm (Agilent) column was used in the Waters Alliance instrument, and 1X PBS pH 7.4 buffer was used for the mobile phase, and the test was performed under the condition of a flow rate of 1.0 ml/min. The results are shown in FIG. 5 . As shown in FIG. 5 , in the case of Hz1, it was confirmed that the aggregate formation was 5%, which was 5.8 times higher than that of Hz2, 0.85, and in the case of Lu007, it was confirmed that the aggregate formation was not as high as 1.1%.

Viscosity measurements were performed as follows. An m-VROC microflow viscometer (RheoSense) was used, and after temperature equilibration at 25 °C, the viscosity was measured three times at each concentration, and the average viscosity (Viscosity, mPa·s) was calculated. After injecting 0.3 ml of a sample into a 0.5 ml Hamilton syringe, the syringe was mounted on a chip enclosure, and the sample was measured after being seated in a thermal jacket. The m-VROC viscometer was equipped with a B05 chip fabricated with a 50 micron channel and measured at multiple flow rates approximately 20%, 40% and 60% of the maximum pressure of the chip. The results are shown in FIG. 6 . As shown in FIG. 6 , it was also confirmed that the viscosity of Hz2 was 12 cP, twice lower than that of Hz1.

Combining the two results, it seems that Hz2 at high concentration has the advantage of low aggregation formation and low viscosity, which can be attributed to the difference in the sequence of the framework part between the two antibodies, and the sequence of Hz2 is responsible for the formation of aggregates and It was thought to contribute to lowering the viscosity.

<Example 10> Evaluation of thermal stability of anti-VEGF antibody through measurement of Tm value

In order to prepare a stable antibody formulation, it is desirable to have high thermal stability. As an evaluation method of stability, evaluation of the heat denaturation intermediate temperature (Tm value) was performed, and in general, it is preferable that the heat denaturation intermediate temperature is high. The thermal stability of each antibody is determined by measuring the fluorescence of the dye by temperature increase as the hydrophobic portion located inside the protein is exposed in the folded state of the protein and a dye having a binding force with the hydrophobic portion is introduced. ) was used. PTS assay was performed using a Protein Thermal Shift Dye Kit (Catalog No. 4461146, Applied Biosystems) according to the manufacturer's method. Specifically, after mixing 5 μg of each antibody with protein thermal shift die and buffer to 20 μl, RT-PCR (C1000 thermal cycler equipped with a CFX96 optical reaction module, Bio-Rad) was performed at 20° C. at 1° C. per 60 seconds. The ROX signal was monitored up to 95 °C with increasing temperature. Based on the melting curve, the melting point (melting temperature, T _m ) of each antibody was determined. Table 2 shows the dissolution points of the antibodies.

Thermal stability test of Hz2 under Hz1 using thermal shift analysis sample T _m (℃) Hz1 74 Hz2 75 Lu007 77 Lucentis-Ig 72 Avastin 71

Looking at the results, it was confirmed that Hz1 and Hz2 were not significantly different in thermal stability and showed a difference of about 1 °C. Compared to the case of Avastin used as a control, it can be said that the thermal stability is good when there is a difference of about 4 ℃. Lu007 showed a difference of more than 5 ℃ compared to the control group, Lucentis-Ig.

<Example 11> Hz2, Hz2-F, Lu007 binding evaluation to FcγR I, II, Ⅲ

In the case of antibody pharmaceuticals whose only purpose is antigen neutralization, effector functions such as ADCC of the Fc region are not required. Binding of the receptor and FcRn is not required.

As the variable region sequence, heavy chain SEQ ID NO: 30 and light chain SEQ ID NO: 31 were used, and mutations of L234A, L235A, P331G were introduced to reduce Fcγ receptor binding to the Fc portion, and mutations of I253A, H310A were introduced to inhibit FcRn binding. An Hz2-F expression vector was prepared. The expression vector was prepared by the method of Example 2, the heavy chain using Hz2-F HC (SEQ ID NO: 32), and the light chain using Hz2 LV, culture and purification were performed according to the method of Example 2. Lu007-F (SEQ ID NO: 38) was prepared in the same manner.

Binding evaluation to FcγRI, II, III was performed as follows. Octet red96E was used, and 100 mM FcγRI (R&D system, 1257-Fc), FcγRII (R&D system, 1597-CF), FcγRIII (R&D system, 1257-Fc) for the anti-penta his biosensor was 5 nm The degree of coating was confirmed. To determine the reactivity of Hz2 (antibody having SEQ ID NO: 30 and SEQ ID NO: 31) and Hz2-F (antibody having SEQ ID NO: 32 and SEQ ID NO: 31) to FcγRI, FcγRII, FcγRIII, the antibody concentration was set to 100 nM The binding force was measured using an analyte. During the measurement, the rpm was set to 100, and the temperature was measured at 30 °C. The sample was diluted using a kinetic buffer (Molecular Device). The antibody solution of each concentration was used as a bound phase for 2 minutes, and then switched to a kinetic buffer to be a dissociated phase for 2 minutes. The binding force between the Fcγ receptor and the antibody is shown in FIG. 7 .

As shown in FIG. 7 , Hz2 appears to normally bind to the Fcγ receptor, and in the case of Hz2-F and Lu007-F, which weakened binding to the Fcγ receptor, it was confirmed that the binding was weak as expected.

<Example 12> Hz2, Hz2-F, Lu007-F binding evaluation to FcRn

Binding evaluation to FcRn (R&D Systems, Inc., 8639-Fc) was performed as follows. Octet red96E was used, and ^{the coating degree of about 5 nm was confirmed by coating an anti-Fab 2 nd} generation (FAB2G) biosensor with 10 μg/ml of Hz2 and Hz2-F antibodies. In order to check the reactivity of FcRn, it was diluted with FcRn binding buffer (100 mM sodium phosphate, 150 mM NaCl, 0.05% Tween20, pH 6.0) so that the FcRn concentration was 400 nM and used as an analyte, and the rpm was set to 100 when measuring. and the temperature was measured at 30 °C. Samples were diluted using FcRn binding buffer. A 400 mM FcRn solution was used as the binding phase for 2 minutes, then switched to the FcRn binding buffer, and the dissociated phase was used for 2 minutes. The binding force of FcRn and antibody is shown in FIG. 8 . As shown in FIG. 8 , Hz2 appears to normally bind to FcRn, and in the case of Hz2-F and Lu007-F, which weakened binding to FcRn, it was confirmed that binding was weak as expected.

<Example 13> Preparation of highly concentrated formulations for Hz1, Hz2-F, and Lu007-F

The most problematic thing when concentrating a protein at a high concentration is a sudden increase in viscosity. In this respect, the importance of viscosity after concentration can be confirmed. It is generally known that the acceptable viscosity range for protein preparations is 20 cP or less. In order to improve the increase in viscosity, which is a problem during such high concentration, a highly concentrated antibody was prepared using three formulations, and the viscosity was measured and compared.

Formulation 1 Formulation 2 Formulation 3 Histidine 10 mM 10 mM 10 mM NaCl 135 mM 135 mM 135 mM Proline - 200 mM 200 mM pH 5.5 5.0 5.5

In the case of Hz1, concentration was performed using an ultrafiltration membrane using Formulations 1 and 2 at concentrations of 150, 180, and 200 mg/ml. In the case of Hz2-F, concentration was performed in the same manner as above at concentrations of 150, 180, 200, 230, and 300 mg/ml. In the case of Lucentis-Ig, concentration was performed to 150, 180, and 200 mg/ml using Formulation 3, and in the case of Lu007, 150, 180, 200, 230, and 300 mg/ml were obtained using Formulations 2 and 3 concentrated to a concentration.

After the concentration was completed, the viscosity measurement was performed as follows. An m-VROC microflow viscometer (RheoSense) was used, and after temperature equilibrium at 25 °C, the viscosity was measured three times at each concentration, and the average viscosity (Viscosity, mPa·s) was calculated. After injecting 0.3 ml of a sample into a 0.5 ml Hamilton syringe, the syringe was mounted on a chip enclosure, and the sample was measured after being seated in a thermal jacket. The m-VROC viscometer was equipped with a B05 chip made with a 50 micron channel and measured at multiple flow rates of approximately 20%, 40% and 60% of the maximum pressure of the chip. The results are shown in Tables 4 and 5.

Viscosity check test after high concentration for Hz1 and Hz2-F Sample name Antibody concentration (mg/ml) Viscosity of Formulation 1 (cP) Viscosity of Formulation 2 (cP) Hz1 150 3.8 2.8 180 5.6 7.2 200 10.0 12 Hz2-F 150 3.0 2.8 180 4.5 3.8 200 5.1 8.0 230 6.9 10.0 300 15.0 20.2

Viscosity confirmation test after high concentration for Lucentis-Ig and Lu007-F Sample name Antibody concentration (mg/ml) Viscosity (CP) of Formulation 2 Viscosity (CP) of Formulation 3 Lucentis-Ig 150 - 4.6 180 - 7.8 200 - 11.0 Lu007-F 150 3.0 3.3 180 4.4 4.2 200 5.3 7.8 230 6.9 9.0

As shown in Table 4, in the case of Hz1, it was confirmed that the viscosity increased as the concentration increased regardless of whether proline was added in the buffer. In the case of Hz2-F, in the case of Formulation 1, it was confirmed that the viscosity increased as the concentration increased, but it was confirmed that even at 300 mg/ml, it showed 15 cP, which is lower than the allowable viscosity of 20 cP. However, the effect of the addition of proline did not appear, and it was confirmed that the viscosity was rather increased. In the case of Hz2-F having a viscosity of 15 cP at a concentration of 300 mg/ml, it can be said that histidine and NaCl are used to prepare an antibody of a high concentration formulation.

In the case of Lu007-F, as shown in Table 5, it was confirmed that the viscosity increased as the concentration increased, regardless of whether proline was added in the buffer, and it was confirmed that the viscosity decreased as the pH decreased. That is, in the case of Lu007-F, it was thought that the increase in viscosity could be further reduced by lowering the pH rather than adding proline.

<110> Medytox Inc. <120> High concentration anti-VEGF antibody formulation and anti-VEGF antibody for use in the same <130> 37-KR-GN-1 <160> 62 <170> KoPatentIn 3.0 <210> 1 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gggcccaggc ggccgagctc gtgmtgaccc agactcca 38 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gggcccaggc ggccgagctc gatmtgaccc agactcca 38 <210> 3 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gggcccaggc ggccgagctc gtgatgaccc agactgaa 38 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tctagaggaa ccacctagga tctccagctc ggtccc 36 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tctagaggaa ccacctttga tttccacatt ggtgcc 36 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tctagaggaa ccacctttga csaccacctc ggtccc 36 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gggcccaggc ggccgagctc gtgctgactc agtcgccctc 40 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tctagaggaa ccacctgtga cggtcagctg ggtccc 36 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cagtcggtgg aggagtccrg g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cagtcggtga aggagtccga g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cagtcgytgg aggagtccgg g 21 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cagsagcagc tgrtggagtc cgg 23 <210> 13 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 actggccggc ctggcctgtg acggtcagct gggtccc 37 <210> 14 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 actggccggc ctggcctgag gagayrgtga ccagggtg 38 <210> 15 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cgagctggag atcctaggtg gttcctctag atct 34 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 caatgtggaa atcaaaggtg gttcctctag atct 34 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cgaggtggts gtcaaaggtg gttcctctag atct 34 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 actcctccac cgactgccca ccaccgcc 28 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 actccttcac cgactgccca ccaccgcc 28 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 actcctccar cgactgccca ccaccgcc 28 <210> 21 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tccaycagct gctsctgccc accaccgcc 29 <210> 22 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gaggaggagg aggaggaggc ggggcccagg cggccgag 38 <210> 23 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gaggaggagg aggaggagcc tggccggcct ggcctgag 38 <210> 24 <211> 116 <212> PRT <213> Oryctolagus cuniculus <400> 24 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Asn Arg Tyr Ser 20 25 30 Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Val Gly Gly Ser Gly Val Thr Tyr Tyr Ala Asn Trp Thr Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Ser 85 90 95 Asp Arg Ser Ala Tyr Ala Pro Asp Ile Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Ile Ser Ser 115 <210> 25 <211> 112 <212> PRT <213> Oryctolagus cuniculus <400> 25 Glu Leu Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr Ser Asn 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Leu Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Val Tyr Asp Asp 85 90 95 Asp Ala Asp Asn Ala Phe Gly Gly Gly Thr Glu Leu Glu Ile Leu Arg 100 105 110 <210> 26 <211> 348 <212> DNA <213> Oryctolagus cuniculus <400> 26 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagtct ctggaatcga cctcaatagg tattcactga cctgggtccg ccaggctcca 120 gggaaggggc tggaatggat cggaatcgtt ggtggtagtg gtgtcacata ctacgcgaac 180 tggacgaaag gccgattcac catctccaaa acctcgacca cggtggatct gaaaatgacc 240 agtctgacaa ccgaggacac ggccacctat ttctgtgcca gaggatctga tcgtagtgct 300 tatgcccctg acatctgggg cccaggcacc ctggtcacca tctcctca 348 <210> 27 <211> 336 <212> DNA <213> Oryctolagus cuniculus <400> 27 gagctcgata tgacccagac tccatcctcc gtgtctgcag ctgtgggagg cacagtcacc 60 atcaattgcc agtccagtca gagtgtttat agtaacaact acttagcctg gtatcagcag 120 aaaccagggc agcctcccaa gctcctgctc taccaggcat ccaaactggc atctggggtc 180 tcatcgcggt tcagcggcag tggatctggg acacatttca ctctcaccat cagcggcgtg 240 cagtgtgacg atgctgccac ttactactgt ctaggcgttt atgatgatga tgctgataat 300 gctttcggcg gagggaccga gctggagatc ctacgt 336 <210> 28 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz1 HV <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Asp Leu Asn Arg Tyr 20 25 30 Ser Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ile Val Gly Gly Ser Gly Val Thr Tyr Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Gly Ser Asp Arg Ser Ala Tyr Ala Pro Asp Ile Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 29 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz1 LV <400> 29 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 Asn Cys Gln Ser Ser Gln Ser Ile Tyr Ser Asn 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu 35 40 45 Leu Leu Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gly Val Tyr Asp Asp 85 90 95 Asp Ala Asp Asn Ala Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys Arg 100 105 110 <210> 30 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 HV <400> 30 Glu 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 Val Ser Gly Ile Asp Leu Asn Arg Tyr 20 25 30 Ser Leu Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Val Gly Gly Ser Gly Val Thr Tyr Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Asp Arg Ser Ala Tyr Ala Pro Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 31 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 LV <400> 31 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Ser Ile Tyr Ser Asn 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Leu Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Val Tyr Asp Asp 85 90 95 Asp Ala Asp Asn Ala Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 32 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2-F Heavy chain <400> 32 Glu 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 Val Ser Gly Ile Asp Leu Asn Arg Tyr 20 25 30 Ser Leu Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Val Gly Gly Ser Gly Val Thr Tyr Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Asp Arg Ser Ala Tyr Ala Pro Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Gly Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 33 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 HV <400> 33 Glu 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 Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Val 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 Lys Tyr Pro His Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 LV <400> 34 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe 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 Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 35 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody F8 HV <400> 35 Glu 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 Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Arg Asp Ala Ser Lys Ser Thr Val 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 Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody E9 HV <400> 36 Glu 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 Val Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Arg Asp Ala Ser Lys Ser Thr Ala 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 Arg Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody H2 HV <400> 37 Glu 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 Val Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Arg Asp Ala Ser Lys Ser Thr Ala 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 Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007-F Heavy chain <400> 38 Glu 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 Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Val 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 Lys Tyr Pro His Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Gly Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 39 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 HFR1 <400> 39 Glu 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 Val Ser Gly Ile Asp Leu Asn 20 25 30 <210> 40 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 HFR2 <400> 40 Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 <210> 41 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 HFR3 <400> 41 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 or Lu007 HFR4 <400> 42 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 43 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 or Lu007 LFR1 <400> 43 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 44 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 LFR2 <400> 44 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu Tyr 1 5 10 15 <210> 45 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 or Lu007 LFR3 <400> 45 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 <210> 46 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz2 or Lu007 LFR4 <400> 46 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz1 or Hz2 HCDR1 <400> 47 Arg Tyr Ser Leu Thr 1 5 <210> 48 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz1 or Hz2 HCDR2 <400> 48 Ile Val Gly Gly Ser Gly Val Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz1 or Hz2 HCDR3 <400> 49 Gly Ser Asp Arg Ser Ala Tyr Ala Pro Asp Ile 1 5 10 <210> 50 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz1 or Hz2 LCDR1 <400> 50 Gln Ser Ser Gln Ser Ile Tyr Ser Asn Asn Tyr Leu Ala 1 5 10 <210> 51 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz1 or Hz2 LCDR2 <400> 51 Gln Ala Ser Lys Leu Ala Ser 1 5 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Hz1 or Hz2 LCDR3 <400> 52 Leu Gly Val Tyr Asp Asp Asp Ala Asp Asn Ala 1 5 10 <210> 53 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 HFR1 <400> 53 Glu 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 Tyr Asp Phe Thr 20 25 30 <210> 54 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 HFR2 <400> 54 Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 <210> 55 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 HFR3 <400> 55 Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 56 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 LFR2 <400> 56 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr 1 5 10 15 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 HCDR1 <400> 57 His Tyr Gly Met Asn 1 5 <210> 58 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 HCDR2 <400> 58 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys 1 5 10 15 Arg <210> 59 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 HCDR3 <400> 59 Tyr Pro His Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 1 5 10 <210> 60 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 LCDR1 <400> 60 Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 61 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 LCDR2 <400> 61 Phe Thr Ser Ser Leu His Ser 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody Lu007 LCDR3 <400> 62 Gln Gln Tyr Ser Thr Val Pro Trp Thr 1 5

Claims (43)

A stable aqueous pharmaceutical composition comprising a humanized antibody against vascular endothelial growth factor (VEGF) as an active ingredient at a concentration of 150 mg/ml or more. The antibody of claim 1 , wherein the antibody comprises a heavy chain variable region having a heavy chain framework having one or more of Kabat numbers 13Q, 37I, 74S, 77T and 105Q compared to human germ-line immunoglobulin; a light chain variable region having a light chain framework having at least one of 22T, 43A, 83F and 100Q; Or a composition comprising a combination thereof. 3. The composition of claim 2, wherein the heavy chain framework further has one or more of 49A and 73D. 4. The antibody according to any one of claims 1 to 3, wherein the antibody is HFR1 of SEQ ID NO: 39 or SEQ ID NO: 53, HFR2 of SEQ ID NO: 40 or SEQ ID NO: 54, HFR3 of SEQ ID NO: 41 or SEQ ID NO: 55, and SEQ ID NO: 42 a heavy chain variable region having one or more heavy chain frameworks of HFR4; a light chain variable region having a light chain framework of at least one of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 44 or SEQ ID NO: 56, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46; Or a composition comprising a combination thereof. 5. The heavy chain of any one of claims 1 to 4, wherein the antibody is a heavy chain of one or more of HCDR1 of SEQ ID NO: 47 or SEQ ID NO: 57, HCDR2 of SEQ ID NO: 48 or SEQ ID NO: 58, and HCDR3 of SEQ ID NO: 49 or SEQ ID NO: 59 a heavy chain variable region having a CDR; a light chain variable region having at least one light chain CDR of LCDR1 of SEQ ID NO: 50 or SEQ ID NO: 60, LCDR2 of SEQ ID NO: 51 or SEQ ID NO: 61, and LCDR3 of SEQ ID NO: 52 or SEQ ID NO: 62; Or a composition comprising a combination thereof. The method according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain variable region of SEQ ID NO: 30; And a composition comprising the light chain variable region of SEQ ID NO: 31. The method according to any one of claims 1 to 6, wherein the antibody comprises a heavy chain variable region of SEQ ID NO: 33; And a composition comprising the light chain variable region of SEQ ID NO: 34. The composition according to any one of claims 1 to 7, wherein the antibody has reduced or eliminated effector function. The composition of any one of claims 1 to 8, wherein the antibody has reduced or eliminated binding ability to one or more of FcγRI, FcγRII, FcγR III, and FcRn. 10. The composition of any one of claims 1-9, wherein the antibody has a mutation in one or more of L234A, L235A, P331G, 1253A and H310A. 11. The composition of any one of claims 1-10, wherein the antibody comprises a heavy chain of SEQ ID NO:32 or SEQ ID NO:38. The composition according to any one of claims 1 to 11, wherein the concentration of the antibody is 150-300 mg/ml. 13. The composition of any one of claims 1-12, wherein the antibody has _{a binding affinity for VEGF of K D} 100 pM or less. 14. The composition according to any one of claims 1 to 13, wherein the antibody has a thermal stability of at least a Tm of 75 °C. 15. A composition according to any one of claims 1 to 14 comprising a buffer and a tonicity agent. 16. The composition of claim 15, wherein the buffer maintains the pH in the range of 5.0 to 7.5. 17. The composition of claim 15 or 16, wherein the buffer is a histidine buffer. 18. The composition of any one of claims 15-17, wherein the buffer has a concentration in the range of 5-50 mM. 19. The composition according to any one of claims 15 to 18, wherein the isotonic agent is a salt. 20. The composition of claim 19, wherein the salt is at least one selected from the group consisting of NaCl, KCl, NaF, KBr, NaBr, Na ₂ SO ₄ , NaSCN, and K ₂ SO _{4 .} 21. The composition of claim 19 or 20, wherein the salt has a concentration in the range of 50-200 mM. 22. The composition of any one of claims 15-21, wherein the isotonic agent is not a sugar or sugar alcohol, or a polyol. 23. The composition of any one of claims 1-22, wherein the composition is free of stabilizers, surfactants, or both. 24. The composition of claim 23, wherein the surfactant comprises polysorbate. 25. The composition of claim 23 or 24, wherein the stabilizing agent comprises one or more amino acids or salts thereof. 26. The composition of any one of claims 22-25 comprising 5-50 mM histidine buffer and 50-200 mM NaCl. 27. The composition according to any one of the preceding claims, having a viscosity of 20 cP or less at 25 °C. 28. The composition according to any one of claims 1 to 27, for preventing or treating an angiogenic disease. 29. The composition of claim 28, wherein the angiogenic disease is an ocular disease. 30. The method of claim 29, wherein the ocular disease is abnormal angiogenesis, choroidal neovascularization (CNV), retinal vascular permeability, retinal edema, diabetic retinopathy, diabetic macular edema, neovascular age-related macular degeneration (AMD), neovascularization (exudative). ) Age-related macular degeneration (AMD), sequelae associated with retinal ischemia, central retinal vein occlusion (CRVO) and posterior segment neovascularization (posterior segment neovascularization) is at least one composition selected from the group consisting of. 31. A composition according to any one of claims 1 to 30 for parenteral administration. 32. The composition of claim 31 for intravitreal injection. 33. The composition according to any one of claims 1 to 32, for administration in one dosing cycle every 3 months or more. 34. A composition according to any one of claims 1 to 33 for administration in a dose ranging from 2 to 30 mg. 35. The composition according to any one of claims 1 to 34, in the form of a solution or a lyophilized powder. 36. The composition of any one of claims 1-35, contained in a vial, ampoule, or prefilled syringe. a heavy chain variable region having a heavy chain framework comprising HFR1 of SEQ ID NO: 39, HFR2 of SEQ ID NO: 40, HFR3 of SEQ ID NO: 41, and HFR4 of SEQ ID NO: 42; And comprising a light chain variable region having a light chain framework consisting of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 44, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46, a humanized anti-VEGF antibody. 38. The method of claim 37, wherein the heavy chain variable region of SEQ ID NO: 30; And an antibody comprising the light chain variable region of SEQ ID NO: 31. 39. The antibody of claim 37 or 38, comprising the heavy chain of SEQ ID NO:32. a heavy chain variable region having a heavy chain framework consisting of HFR1 of SEQ ID NO: 53, HFR2 of SEQ ID NO: 54, HFR3 of SEQ ID NO: 55, and HFR4 of SEQ ID NO: 42; And comprising a light chain variable region having a light chain framework consisting of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 56, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46, a humanized anti-VEGF antibody. 41. The method of claim 40, wherein the heavy chain variable region of SEQ ID NO: 33; And an antibody comprising the light chain variable region of SEQ ID NO: 34. 42. The antibody of claim 40 or 41 comprising the heavy chain of SEQ ID NO:38. heavy chain variable region of SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37; And comprising the light chain variable region of SEQ ID NO: 34, a humanized anti-VEGF antibody.

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