CN115109154A - Antibody targeting CLDN18.2 or antigen binding fragment thereof and application thereof - Google Patents

Antibody targeting CLDN18.2 or antigen binding fragment thereof and application thereof Download PDF

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CN115109154A
CN115109154A CN202110287671.0A CN202110287671A CN115109154A CN 115109154 A CN115109154 A CN 115109154A CN 202110287671 A CN202110287671 A CN 202110287671A CN 115109154 A CN115109154 A CN 115109154A
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amino acid
acid sequence
antibody
ser
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郎国竣
闫鑫甜
谭永聪
刘婵娟
孔超
闫闰
胡宇豪
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Sanyou Biopharmaceuticals Co Ltd
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Sanyou Biopharmaceuticals Co Ltd
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Priority to PCT/CN2022/081181 priority patent/WO2022194201A1/en
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Abstract

The invention discloses an antibody targeting CLDN18.2 or an antigen binding fragment thereof and application thereof. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, the sequence information of which is detailed in the present invention. The binding capacity of the antibody prepared by the invention and cells such as hLDN 18.2-HEK293T cells, hLDN 18.2-KATOIII cells and the like is equivalent to or better than that of a control antibody IMAB 362; and the ADCC and CDC equivalent on hLDN 18.2-KATOIII cells, hLDN 18.2-HEK293T or hLDN 18.2-HEK293 cells is obviously better than that of a control antibody IMAB 362.

Description

Antibody targeting CLDN18.2 or antigen binding fragment thereof and application thereof
Technical Field
The invention belongs to the field of antibodies, and particularly relates to a CLDN18.2 binding molecule, in particular to an antibody targeting CLDN18.2 or an antigen binding fragment thereof and application thereof.
Background
Cell connection is an intercellular connection structure, and is an important basis for the mutual connection and synergy between adjacent cells in a multicellular organism. In general, there are four types of junctions, tight junctions, adhesive junctions, gap junctions and desmosomes/hemidesmosomes, in animal cells.
Tight junctions, also known as closed junctions, are structures formed between endothelial cells or epithelial cells that prevent diffusion of materials between tissues from the intercellular space and thus can only enter the cell by active transport. The tightly connected structure is formed by dozens of Claudin proteins through protein interaction in and among cells, and the expression of the proteins has certain tissue specificity. CLDN18 is an important protein among Claudin proteins present in tight junctions.
CLDN18 is a membrane protein with a tetraspanin domain, containing two extracellular domains. Two CLDN18 variants have been found in humans, CLDN18.1 and CLDN18.2, respectively. The two are distributed in different tissues, the former is mainly expressed in lung epithelial cells, the latter is specifically expressed in stomach epithelial cells and is not expressed in stomach stem cells. On the protein sequence, CLDN18.1 and CLDN18.2 have very high sequence similarity, the main difference between them is at the N-terminus, and there is only 8 amino acid difference between them in the first extracellular domain; the C-terminal sequence is identical except for the N-terminal.
Antibody therapy is becoming one of the most promising approaches to the treatment of cancer. CLDN18.2 has now become a very potential target for antibody drug action due to its high expression in a variety of tumor tissues, such as non-small cell lung (25%), Gastric (70%), pancreatic (50%) and esophageal (30%), but little in normal tissues (Kumar, v., et al (2018). "emulsifying therapeutics in the Management of Advanced-Stage targeted cancer. However, since the sequence similarity of CLDN18.1 and CLDN18.2 is very high, it is extremely difficult to develop an antibody directed against CLDN18.2 only and not against CLDN 18.1.
Although there is currently a clinical study of monoclonal antibody drug IMAB362 targeted to CLDN18.2 target developed by Ganymed, germany, there is still an urgent need to continue to develop antibodies targeted to CLDN18.2 target as a therapeutic.
Disclosure of Invention
The invention aims to overcome the defect of lack of target CLDN18.2 target antibodies in the prior art, and provides an antibody targeting CLDN18.2 or an antigen binding fragment thereof and application thereof. In particular, a novel antibody recognizing specifically CLDN18.2 and having more excellent effects was developed.
The invention mainly solves the technical problems through the following technical scheme.
The first aspect of the invention relates to an antibody or antigen-binding fragment thereof targeting CLDN18.2 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein:
the HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 5 or a variant 1 thereof, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 6, the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 7 or a variant 2 thereof, the LCDR1 comprises an amino acid sequence shown as SEQ ID NO. 8 or a variant 3 thereof, the LCDR2 comprises an amino acid sequence shown as SEQ ID NO. 9 or SEQ ID NO. 46 or a variant 4 thereof, and the LCDR3 comprises an amino acid sequence shown as SEQ ID NO. 10 or a variant 5 thereof;
the above variant is a substitution, deletion or addition of 1, 2 or 3 amino acids based on the original sequence, and an antibody or antigen-binding fragment thereof comprising the variant retains at least the binding ability to CLDN 18.2.
Wherein, the mutation of the variant 1 is the amino acid substitution of individual positions of the amino acid sequence shown in SEQ ID NO. 5; preferably at least at position 2 of the amino acid sequence shown in SEQ ID NO. 5.
Mutations in variants 2-5 are also amino acid substitutions in the original sequence, specifically:
the mutation of variant 2 preferably occurs at least at positions 14 and/or 15 of the amino acid sequence shown in SEQ ID NO. 7.
The mutation of variant 3 preferably occurs at least at one or more of positions 7, 10 and 12 of the amino acid sequence shown in SEQ ID NO. 8.
The mutation of variant 4 preferably occurs at least at position 4 and/or 5 of the amino acid sequence shown in SEQ ID NO. 46.
The mutation of variant 5 preferably occurs at least at one or more of positions 5, 8 and 9 of the amino acid sequence shown in SEQ ID NO. 10.
In a preferred embodiment of the present invention, the 2 nd position in the variant 1 is preferably mutated to R, i.e., the amino acid sequence thereof is shown in SEQ ID NO. 31.
In the variant 2, the 14 th position preferred mutation is N or P, and the 15 th position preferred mutation is L.
In the variant 3, the 7 th site is preferably mutated into S, the 10 th site is preferably mutated into R, and the 12 th site is preferably mutated into R.
In the variant 4, the 4 th position is preferably mutated to E, and the 5 th position is preferably mutated to R.
In the variant 5, the 5 th site is preferably mutated into W or F, the 8 th site is preferably mutated into L, and the 9 th site is preferably mutated into V or S.
In a preferred embodiment of the invention, the amino acid sequence of variant 2 is shown in SEQ ID NO 32, 33 or 45.
In another preferred embodiment of the present invention, the amino acid sequence of said variant 3 is as set forth in SEQ ID NO 23, 39 or 40;
in another preferred embodiment of the present invention, the amino acid sequence of said variant 4 is as shown in SEQ ID NO. 24 or 41;
in another preferred embodiment of the invention, the amino acid sequence of said variant 5 is as shown in SEQ ID NO 25, 26, 27, 42 or 43.
In a more preferred embodiment of the present invention, the amino acid sequence of HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 7.
In another more preferred embodiment of the present invention, the amino acid sequence of HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 45.
In another more preferred embodiment of the present invention, the amino acid sequence of HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 33.
In another more preferred embodiment of the present invention, the amino acid sequence of HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 32.
In another more preferred embodiment of the present invention, the amino acid sequence of HCDR1 is shown as SEQ ID NO. 31, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 33.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 8, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 10.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 23, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 46, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 25.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 23, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 46, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 26.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 24, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 25.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 23, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 46, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 27.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 23, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 46, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 42.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 39, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 46, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 43.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 40, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 46, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 10.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 8, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 41, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 10.
In another more preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 8, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 46, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 42.
The framework regions of the antibodies or antigen-binding fragments thereof of the present invention are preferably human or murine framework regions.
The amino acid sequence of the heavy chain variable region is preferably shown as SEQ ID NO. 3, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30 or SEQ ID NO. 44.
The amino acid sequence of the light chain variable region is preferably shown as SEQ ID NO. 4, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37 or SEQ ID NO. 38.
In a specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 7, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 9, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 10.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 7, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 40, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 10.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 7, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 41, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 10.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 7, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 42.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 32, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 25.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 25.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 26.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 24, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 25.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 27.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 42.
In another specific embodiment of the invention, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 45, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 39, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 43.
In a most preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 3 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 4 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 15 or a variant thereof and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 18 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 15 or a variant thereof and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 17 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 16 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 18 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 16 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 17 or a variant thereof.
In another preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 28 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 19 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 29 or a variant thereof and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 19 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 30 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 20 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 30 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 21 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 30 or a variant thereof and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 22 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 30 or a variant thereof and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 34 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 44 or a variant thereof and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 35 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 16 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 36 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 16 or a variant thereof and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 37 or a variant thereof.
In another preferred embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 16 or a variant thereof and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 38 or a variant thereof.
In the above-described preferred embodiments, the variant retains at least the function of the pre-mutation sequence and the variant has at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identity to the pre-mutation sequence.
In the present invention, the amino acid sequences of the CDRs listed above are all shown in accordance with the AbM definition rule (the sequences shown in the claims of the present invention are also shown in accordance with the AbM definition rule). However, it is well known to those skilled in the art that CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia (Chothia et Al (1989) Nature 342: 877-Buffer 883, Al-Lazikani et Al, "Standard constraints for the structural organization of immunology", Journal of Molecular Biology,273, 927-Buffer 948(1997)), Kabat (Kabat et Al, Sequences of Proteins of Immunological Interest, 4 th edition, U.S. Department of Health and service, National instruments of Health (1987)), Abstract of Molecular Interest (balance of science), general connectivity of Molecular Interest, and the use of the general crystal GT/. for the purpose of clustering. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are understood to encompass complementarity determining regions as defined by any of the above-described known schemes described by the present invention. Although the scope of the claims of the present invention is based on the sequence shown in the definition rule of AbM, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of the present invention.
Antibody CDR definition method
Figure BDA0002981156570000071
Figure BDA0002981156570000081
Wherein Laa-Lbb can refer to the amino acid sequence from aa to bb, starting from the N-terminus of the antibody light chain; Haa-Hbb may refer to the amino acid sequence from aa to bb, starting from the N-terminus of the heavy chain of the antibody. For example, L24-L34 may refer to the amino acid sequence from position 24 to position 34, beginning at the N-terminus of the antibody light chain, according to the Chothia coding rules; H26-H32 can refer to the amino acid sequence from position 26 to position 32, beginning at the N-terminus of the antibody heavy chain, according to the Chothia coding rules.
Thus, where reference is made to an antibody defined with a particular CDR sequence as defined herein, the scope of the antibody also encompasses an antibody whose variable region sequences comprise the particular CDR sequence but whose claimed CDR boundaries differ from the particular CDR boundaries as defined herein due to the application of different protocols (e.g., different assignment system rules or combinations).
The antibody of the invention preferably further comprises an antibody heavy chain constant region and an antibody light chain constant region; preferably, the heavy chain constant region is derived from a human antibody heavy chain or a variant thereof and the light chain constant region is derived from a human antibody kappa chain or lambda chain or a variant thereof.
The antibody of the present invention may be a full-length antibody, Fab ', F (ab') 2 Fv, bispecific antibodyAn antibody or a multispecific antibody, or a monoclonal antibody or a polyclonal antibody prepared from the above antibody. The Fv is preferably a scFv.
In the present invention, the term "full length antibody" is used interchangeably to refer to a glycoprotein comprising at least two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region consists of one domain CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as lambda or kappa. Immunoglobulins comprising alpha, delta, epsilon, gamma and mu heavy chains are classified as immunoglobulins (Ig) a, IgD, IgE, IgG and IgM. The complete antibody formed a "Y" shape. The stem of Y consists of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have a constant region consisting of three tandem (in-line) Ig domains, and a hinge region for increased flexibility; heavy chains mu and epsilon have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH 2 domain" and the "CH 3 domain", respectively. Each arm of Y comprises the variable region and the first constant region of a single heavy chain joined to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.
In the present invention, the "Fab fragment" consists of one light chain and one heavy chain of CH1 and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains. A "Fab ' fragment" contains a light chain and a portion of a heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form F (ab ') 2 A molecule. "F (ab') 2 Fragment "contains twoA strip light chain and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus F (ab') 2 The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The term "Fv" means an antibody fragment consisting of the VL and VH domains directed to a single arm of an antibody, but lacking a constant region.
In the present invention, the scFv (single chain antibody) can be a single chain antibody conventional in the art, and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids. Wherein the VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain [ see, e.g., Bird et al, Science 242:423-]. Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art joints are made of repeating G 4 S amino acid sequence or a variant thereof. For example, a polypeptide having an amino acid sequence (G) 4 S) 4 Or (G) 4 S) 3 Linkers, but variants thereof may also be used.
The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; an antibody having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently, and the like.
The antibodies of the invention include monoclonal antibodies. The monoclonal antibody or mAb or Ab of the present invention refers to an antibody obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines.
A second aspect of the invention relates to an isolated nucleic acid encoding an antibody targeting CLDN18.2 or an antigen-binding fragment thereof according to the first aspect of the invention.
A third aspect of the invention relates to a recombinant expression vector comprising an isolated nucleic acid as described in the second aspect of the invention. Preferably, the recombinant expression vector is a plasmid, cosmid, phage, or viral vector, preferably a retroviral vector, lentiviral vector, adenoviral vector, or adeno-associated viral vector.
The fourth aspect of the present invention relates to a transformant comprising the recombinant expression vector according to the third aspect of the present invention in a host cell; preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of yeast cells, mammalian cells (e.g., 293 cells or CHO cells) or other cells suitable for the production of antibodies or antigen binding fragments thereof. Once an expression vector or DNA sequence has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and mammalian host cell used based on the present specification and methods known in the art. Alternatively, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics), or heavy metal (e.g., copper) resistance, etc., to the auxotrophic host. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.
A fifth aspect of the invention relates to a chimeric antigen receptor comprising an antibody targeting CLDN18.2 or an antigen-binding fragment thereof according to the first aspect of the invention.
A sixth aspect of the invention relates to a genetically modified cell comprising a chimeric antigen receptor according to the fifth aspect of the invention; preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably immune cells such as T cells, or NK cells.
The seventh aspect of the present invention relates to a method for producing an antibody having the target CLDN18.2 or an antigen-binding fragment thereof, comprising culturing the transformant according to the fourth aspect, and obtaining the antibody having the target CLDN18.2 or the antigen-binding fragment thereof from the culture.
An eighth aspect of the invention relates to an antibody drug conjugate comprising a cytotoxic agent, and an antibody or antigen-binding fragment thereof having the target CLDN18.2 according to the first aspect; preferably, the cytotoxic agent is MMAF or MMAE.
A ninth aspect of the invention relates to a pharmaceutical composition comprising an antibody having the target CLDN18.2 of the first aspect or an antigen-binding fragment thereof and/or an antibody drug conjugate of the eighth aspect, and a pharmaceutically acceptable carrier;
preferably, the pharmaceutical composition further comprises one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.
In some embodiments, the pharmaceutical composition or pharmaceutical formulation of the invention comprises a suitable pharmaceutically acceptable carrier, e.g. a pharmaceutical excipient, such as a pharmaceutical carrier, pharmaceutical excipient, known in the art, including a buffer. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of Pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, Pharmaceutical Press, London, Chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin. A Pharmaceutical formulation or Pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention having the desired purity may be prepared by mixing the antibody or antigen-binding fragment thereof with one or more optional Pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators and the like. The active ingredients are suitably present in combination in an amount effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibodies, or antigen-binding fragments thereof, of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
A tenth aspect of the present invention relates to the use of an antibody targeting CLDN18.2 or an antigen-binding fragment thereof according to the first aspect, an antibody drug conjugate according to the eighth aspect, and/or a pharmaceutical composition according to the ninth aspect of the present invention for the preparation of a medicament for the diagnosis, prevention and/or treatment of a tumor; preferably, the tumor is a CLDN18.2 positive tumor; more preferably, the tumor is gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and/or leukemia.
An eleventh aspect of the invention relates to a kit comprising an antibody having a CLDN18.2 target or an antigen-binding fragment thereof according to the first aspect, a chimeric antigen receptor according to the fifth aspect, a genetically modified cell according to the sixth aspect, or an antibody drug conjugate according to the eighth aspect or a pharmaceutical composition according to the ninth aspect;
preferably, the kit further comprises (i) a means for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
A twelfth aspect of the invention relates to a kit of parts comprising kit a and kit B, wherein:
the kit a contains an antibody targeting CLDN18.2 or an antigen-binding fragment thereof of the first aspect, a chimeric antigen receptor of the fifth aspect, a genetically modified cell of the sixth aspect, an antibody drug conjugate of the eighth aspect and/or a pharmaceutical composition of the ninth aspect;
the kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising said other anti-tumor antibodies and/or one or more of the group consisting of hormonal agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.
A thirteenth aspect of the invention relates to a method of diagnosing, treating and/or preventing a CLDN18.2 mediated disease or disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof targeting CLDN18.2 of the first aspect of the invention, a chimeric antigen receptor of the fifth aspect, an antibody drug conjugate of the sixth aspect or a pharmaceutical composition of the eighth aspect, or treating a patient in need thereof using a kit of parts of the twelfth aspect.
Wherein the disease or disorder may be a tumor, preferably a CLDN18.2 positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and/or leukemia.
A fourteenth aspect of the present invention relates to a method of immunodetection or determination of CLDN18.2, comprising using the antibody or antigen-binding fragment thereof targeting CLDN18.2 of the first aspect, the chimeric antigen receptor of the fifth aspect, the antibody drug conjugate of the eighth aspect, or the pharmaceutical composition of the ninth aspect; preferably, the detection is for non-diagnostic and/or therapeutic purposes.
A fifteenth aspect of the present invention relates to a combination therapy comprising administering to a patient in need thereof an antibody targeting CLDN18.2 or an antigen-binding fragment thereof according to the first aspect, a chimeric antigen receptor according to the fifth aspect, an antibody drug conjugate according to the eighth aspect or a pharmaceutical composition according to the ninth aspect, respectively, and a second therapeutic agent; the second therapeutic agent preferably comprises an additional anti-tumor antibody or a pharmaceutical composition comprising the additional anti-tumor antibody, and/or one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.
Further, explanations of other terms used in the present invention are specifically as follows.
The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. The construction of chimeric antibody includes the first establishing hybridoma secreting mouse-derived specific monoclonal antibody, the subsequent cloning of variable region gene from mouse hybridoma cell, the subsequent cloning of human antibody constant region gene, connecting the mouse variable region gene and human constant region gene into chimeric gene, inserting the chimeric gene into human carrier, and final expressing the chimeric antibody molecule in eukaryotic industrial system or prokaryotic industrial system. In a preferred embodiment of the invention, the antibody light chain targeting a CLDN18.2 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the CLDN 18.2-targeted chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof. The constant region of the human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG2 or IgG4 heavy chain constant region, or IgG4 which is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.
"humanized antibody" refers to a class of engineered antibodies having CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin portion of the humanized antibody being derived from one (or more) human immunoglobulin(s). Furthermore, framework support residues may be altered to preserve binding affinity (see, e.g., Queen et al, Proc. Natl. Acad. Sci. USA,86:10029-10032(1989), Hodgson et al, Bio/Technology,9:421 (1991)). Suitable human acceptor antibodies may be antibodies selected from conventional databases, such as the Los Alamos database and the Swiss protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (based on amino acids) to the framework regions of the donor antibody may be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies that provide light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains need not be derived from the same acceptor antibody.
As known in the art, "polynucleotide" or "nucleic acid" used interchangeably in the present invention refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a strand by a DNA or RNA polymerase.
The calculation of sequence identity between sequences was performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. Sequence comparisons between two sequences and calculation of percent identity can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J.mol.biol.48: 444-. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com), using the NWSgapdna. CMP matrix and GAP weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix using a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the PAM120 weighted residue table, gap length penalty 12, gap penalty 4) using the e.meyers and w.miller algorithms that have been incorporated into the ALIGN program (version 2.0) ((1989) cabaos, 4: 11-17). Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
As used herein, "vector" means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
The term "host cell" in the context of the present invention can include cells into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. The invention includes mutant progeny that have the same function or biological activity as the cell selected or selected for in the originally transformed cell.
As used herein, the term "effective amount" means an amount of a drug or pharmaceutical agent that elicits the biological or pharmacological response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a decrease in the rate of progression of a disease or condition, as compared to a corresponding subject not receiving that amount. The term also includes within its scope an amount effective to enhance normal physiological function.
In some embodiments, the amino acid change can be an addition, deletion, or substitution of an amino acid, e.g., the amino acid change is a conservative amino acid substitution. In some embodiments, the amino acid change does not occur in a CDR region.
In some embodiments of the invention, the amino acid changes described herein comprise amino acid substitutions, insertions or deletions. In some embodiments, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions are those where one amino acid is substituted with another within the same class, for example where one acidic amino acid is substituted with another acidic amino acid, one basic amino acid is substituted with another basic amino acid, or one neutral amino acid is substituted with another neutral amino acid.
It should be noted that: the numerical values after the modification in the present invention such as "1", "2", etc. have no essential meanings, and are only to distinguish the same terms.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the binding capacity of the antibody prepared by the invention and cells such as hLDN 18.2-HEK293T cells and hLDN 18.2-KATOIII cells is equivalent to or better than that of a control antibody IMAB 362; and the ADCC and CDC killing effects on hLDN 18.2-KATOIII cells are obviously better than those of a control antibody IMAB362, and the CDC effects on hLDN 18.2-HEK293T or hLDN 18.2-HEK293 cells are also obviously better than those of the control antibody IMAB 362. Wherein the ADCC effect of the antibody on hCDN18.2-HEK 293 cells after the exemplary modification of the invention is better than that of other control antibodies ASK589-M5, h1901-11 and hu8E 5-2I. In addition, in the non-specific cell binding experiment, the binding capacity of the antibody of the present invention to cells other than the target cells was weaker than that of the control antibody IMAB 362.
Drawings
Figure 1 shows binding of Phage (surface display of corresponding Fab fragments) from partial clones (clone No. 262 and clone No. 188) of the mouse immune pool to hcldn18.1-HEK293 cells and hcldn18.2-HEK293T cells, respectively.
Fig. 2a, 2b and 2c show the binding of human murine chimeric antibody 262 of the mouse immune repertoire to cells overexpressing human CLDN18.2 or overexpressing human CLDN 18.1. Wherein FIG. 2a shows the binding of human murine chimeric antibody 262 to hCLDN18.2-HEK293T cells; FIG. 2b shows the binding of human murine chimeric antibody 262 to hCLDN18.2-KATOIII cells; FIG. 2c shows the binding of human murine chimeric antibody 262 to hCLDN18.1-HEK293 cells.
Fig. 3a, 3b and 3c show the binding of a series of humanized antibodies derived from the human murine chimeric antibody 262 after humanization to cells overexpressing human CLDN18.2 or overexpressing human CLDN 18.1. Wherein FIG. 3a shows the binding of humanized antibodies 262-H1L1, 262-H1L2, 262-H2L1, 262-H2L2 to hCLDN18.2-HEK293T cells; FIG. 3b shows the binding of humanized antibodies 262-H1L2, 262-H2L2 to hCLDN18.2-KATOIII cells; FIG. 3c shows binding of humanized antibodies 262-H1L1, 262-H1L2, 262-H2L1, 262-H2L2 to hCLDNN 18.1-HEK293 cells.
Fig. 4a, 4b show the CDC effect of human murine chimeric antibody 262 on cells overexpressing human CLDN 18.2. Wherein, FIG. 4a shows CDC effect of human murine chimeric antibody 262 on hCLDN18.2-HEK293T cells; FIG. 4b shows the CDC effect of human murine chimeric antibody 262 on hCLDN18.2-KATOIII cells.
Fig. 5a, 5b show the CDC effect of humanized antibody 262-H1L2 on cells overexpressing human CLDN 18.2. Wherein, FIG. 5a shows the CDC effect of humanized antibody 262-H1L2 on hCLDNN 18.2-HEK293T cells; FIG. 5b shows the CDC effect of humanized antibody 262-H1L2 on hCLDNN 18.2-KATOIII cells.
Fig. 6a, 6b show the CDC effect of humanized antibody 262-H2L2 on cells overexpressing human CLDN 18.2. Wherein, figure 6a shows the CDC effect of humanized antibody 262-H2L2 on hcldn18.2-HEK293T cells; FIG. 6b shows the CDC effect of humanized antibody 262-H2L2 on hCLDNN 18.2-KATOIII cells.
FIG. 7 shows the ADCC effect of human murine chimeric antibody 262 on hCLDN18.2-KATOIII cells.
FIG. 8 shows the ADCC effect of humanized antibody 262-H1L2 on hCLDNN 18.2-KATOIII cells.
FIG. 9 shows the ADCC effect of humanized antibody 262-H2L2 on hCLDNN 18.2-KATOIII cells.
Fig. 10a, 10b show binding of humanized antibody 262-H2L2 to cells overexpressing human CLDN18.2 or overexpressing human CLDN18.1 after a series of modifications of the antibody obtained after antibody engineering. Wherein figure 10a shows binding of the engineered antibody to hcldn18.2-HEK293 cells; FIG. 10b shows binding of the engineered antibody to hLDN 18.1-HEK293 cells.
FIG. 11 shows non-specific binding of engineered antibodies 262-H2L2-7, 262-H2L2-11, 262-H2L2-23, 262-H2L2-24, 262-H2L2-53 to CHO-K cells.
Fig. 12a, 12b, 12c, 12d, 12e show the CDC effect of the engineered antibodies on HEK293 cells overexpressing human CLDN 18.2. Wherein, FIGS. 12a to 12e respectively show CDC effects of the engineered antibodies 262-H2L2-7, 262-H2L2-11, 262-H2L2-23, 262-H2L2-24, 262-H2L2-53 on hCLDNN 18.2-HEK293 cells.
FIGS. 13a, 13b, 13c, 13d show the ADCC effect of the engineered antibodies 262-H2L2-7, 262-H2L2-23 on hCLDN18.2-HEK293 cells. Wherein, FIGS. 13a and 13b show the ADCC effect of the engineered antibody 262-H2L2-7 on hCDLN 18.2-HEK293 cells; FIGS. 13c and 13d show the ADCC effect of the engineered antibody 262-H2L2-23 on hCDN18.2-HEK 293 cells.
FIG. 14 shows the inhibition of tumor growth in mice by the engineered antibodies 262-H2L2-2, 262-H2L2-9, 262-H2L2-14, 262-H2L2-19, 262-H2L 2-20.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 preparation of overexpressing cell lines
1.1 flow assay of HEK293T cell line overexpressing human CLDN18.2
A cell line of HEK293T (hCDLN 18.2-HEK293T) overexpressing human CLDN18.2 was purchased from Congyuan Bingchu under the accession number KC-0986. hCL was performed using IMAB362 antibody (expressed and purified according to the sequence information of the heavy chain SEQ ID NO:17 and the light chain SEQ ID NO:24 as disclosed in WO2016165762A 1)DN18.2-HEK 293T. Take 1X 10 5 Individual cells were centrifuged at low speed (300g) to remove supernatant. The cells at the bottom of the centrifuge tube were rinsed once with a prepared FACS buffer (1 XPBS buffer containing 2% FBS by volume), and then 12.5. mu.g/mL of antibody IMAB362 was added to the rinsed cells, followed by incubation at 4 ℃ for 1 hour, and then rinsed three times with the above FACS buffer, and 0.5. mu.g of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) was added, followed by incubation at 4 ℃ for 1 hour. Thereafter, the cells were rinsed three times with FACS buffer, and 200. mu.L of FACS buffer was added to the cells to resuspend the cells, and finally detected by a flow cytometer (Beckman, Cytoflex, AOO-1-1102). The detection result shows that the surface of the commercial hCDLN 18.2-HEK293T highly expresses human CLDN18.2 protein.
1.2 construction and flow identification of HEK293 cell line overexpressing human CLDN18.1
Construction of HEK293 cell line overexpressing human CLDN18.1 (hereinafter, hCLDN18.1-HEK 293): the nucleic acid sequence of full-length human CLDN18.1(SEQ ID NO:1) was constructed onto the pLVX-puro plasmid (Clontech, Cat # 632164). The resulting plasmid was then passed through an electrotransfer instrument (Invitrogen, Neon) TM (transformation System, MP922947) electrotransformation into HEK293 cells
Figure BDA0002981156570000171
CRL-1573 TM ) In (1). After the electrotransformation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and containing no antibiotics, respectively, and then the cells were seeded into 10X 10cm cell culture dishes and cultured for 48 hours, and then the cells were dispensed into 96-well cell culture plates at an average density of 0.5/well, puromycin (Gibco, A111138-03) was added at a final concentration of 2. mu.g/mL as a selection pressure, the clonal growth of the cell strain was observed for about 2 weeks, and the cell strain forming the clone was picked up and identified.
Flow assay of hCLDN18.1-HEK 293: cells were fixed and disrupted as described in the cell disruption kit (eBioscience, 88-8824-00), and anti-CLDN 18 antibody (Abcam, ab203563) was added and incubated at 4 ℃ for 1 hour. Rinsing with membrane-breaking buffer solutionAfter three passes Alexa was added
Figure BDA0002981156570000172
488 fluorescence-labeled donkey anti-rabbit IgG H&L (Abcam, ab150073) 0.5. mu.g, incubated at 4 ℃ for 1 hour and finally detected by flow cytometry (Beckman, CytofLAAO-1-1102). Also using the method of 1.1 above, hCLDN18.1-HEK293 was excluded from expressing CLDN18.2 using the IMAB362 antibody. The detection result shows that the hLDN 18.1-HEK293 surface highly expresses human CLDN 18.1.
1.3 construction and identification of KATOIII cells overexpressing human CLDN18.2
The gastric cancer cell strain KATOIII (hLDN 18.2-KATOIII) over-expressing human CLDN18.2 is constructed by lentivirus transfection and identified by antibody IMAB 362. The specific method comprises the following steps: taking 5X 10 4 Well-conditioned human gastric cancer cells (katoii,
Figure BDA0002981156570000173
HTB-103 TM ) And the ratio of 30: 1, packaged lentivirus containing the human CLDN18.2 sequence (SEQ ID NO:2), mixed well, then IMDM complete medium (Gibco, 12440061) containing 8 μ g/mL Polybrene (Polybrene, Sigma, 107689) was added, mixed well, CO 5% concentration at 37 ℃, CO 2 The constant temperature incubator of (1) was incubated for 20 hours, the medium was removed, fresh IMDM complete medium was replaced and incubation was continued for 24 hours, then transfected KATOIII cells were seeded into 96-well plates at an average density of 0.5 cells/well and puromycin was added to a final concentration of 2. mu.g/mL for resistance pressure screening at 37 ℃ with 5% CO 2 Culturing for 2-3 weeks in a constant temperature incubator, and selecting clones for identification.
The cell lines subjected to resistance selection were identified by flow assay using antibody IMAB362 according to the method described in 1.1 above. The results of flow assay showed that KATOIII cells not transfected with CLDN18.2 were hardly recognized by antibody IMAB362, indicating that this KATOIII cell strain hardly expressed CLDN18.2, and hcldn18.2-KATOIII transfected by lentivirus could be recognized by antibody IMAB362, and this cell strain successfully highly expressed CLDN18.2 on the surface.
1.4 construction and identification of HEK293 cell line over-expressing human CLDN18.2
Construction of a HEK293 cell line overexpressing human CLDN18.2 (hereinafter hCDLN 18.2-HEK 293): the nucleic acid sequence of full-length human CLDN18.2(SEQ ID NO:2) was constructed onto the pLVX-puro plasmid (Clontech, Cat #632164) and the remaining steps were referenced to example 1.2. The detection result proves that the hCLDN18.2-HEK293 surface highly expresses human CLDN 18.2.
1.5 construction and characterization of CD16a (F158) -NF-AT-jurkat cell line
First, an NF-AT-Jurkat cell line was constructed, and pGL4.30 plasmid (promega, # E8481) containing an NF-AT-re nucleic acid sequence was electrically transformed into Jurkat cells by an electrotransfer instrument (Invitrogen, Neon transfer System, MP922947) ((R))
Figure BDA0002981156570000181
CRL-1573 TM ) In (1). After the electrotransformation, the clone growth of the cell line was observed for about 2-3 weeks using Hygromycin B (source culture, S160J7) at a final concentration of 500 μ g/mL as a selection pressure, and the cell line forming the clone was picked up and identified. After the NF-AT-Jurkat cell strain is qualified, the ratio of 20: 1 (MoI) into a packaged lentivirus containing the sequence CD16a (F158) (UniProtKB-P08637, F phenylalanine at amino acid 158), was subjected to resistance stress screening with the addition of puromycin at a final concentration of 2. mu.g/mL, and the mixture was subjected to 5% CO concentration at 37 ℃ under pressure 2 Cultured in an incubator for 2-3 weeks, and clones are picked for identification, thereby finally successfully obtaining the CD16a (F158) -NF-AT-jurkat cell strain.
Example 2 animal immunization
2.1 immunization protocol
MRL mice (Shanghai Ling Biotech Co., Ltd., female, 6-8 weeks, n. times.4) were immunized by subcutaneous and intraperitoneal injections with hLDN 18.2-HEK293T as the immunogen. Immunizations were performed every two weeks for a total of 4 immunizations. Finally, hLDN 18.2-HEK293T cells were immunized once more.
2.2 detection of serum antibody titres in mice after immunization
hCDNN 18.2-HEK293T cells, hCDNN 18.1-HEK293 cells and HEK293T cells were taken, and 500g of the supernatant was centrifuged at low speed. The non-immunized negative and post-immune sera were diluted with FBS on another 96-well dilution plate and diluted 50-fold over 4 gradients (1/50, 1/250, 1/1250, 1/6250). The cells at the bottom of the centrifuge tube were rinsed once with a prepared FACS buffer (1 XPBS buffer containing 2% FBS by volume), and then diluted immune serum was added to the rinsed cells, followed by incubation at 4 ℃ for 1 hour, and then rinsed three times with the above FACS buffer, and 0.5. mu.g of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) was added, followed by incubation at 4 ℃ for 1 hour. Thereafter, the cells were rinsed three times with FACS buffer, and 200. mu.L of FACS buffer was added to the cells to resuspend the cells, and finally detected by a flow cytometer (Beckman, Cytoflex, AOO-1-1102). The detection result shows that the serum of the mouse has the antibody with high recognition of CLDN18.2 and low recognition of CLDN 18.1.
Example 3 mouse immune library construction and screening
3.1 phage display antibody Gene library construction
After immunization, mice were treated according to the standard protocol of euthanasia. Collecting spleen cells from mice, grinding and filtering, adding 1mL TRIzol TM Reagent (Thermo Fisher, 15596026) lysed spleen cells, total RNA was extracted by phenol chloroform method, and the extracted RNA was reverse transcribed into cDNA by reverse transcription kit (TaKaRa, 6210A). Then cDNA is used as a PCR template, and specific primers of a murine antibody sequence are adopted to respectively amplify light chain genes and heavy chain genes of the antibody. The PCR product was digested with NcoI and NotI to obtain an antibody gene fragment, which was inserted into a phage display vector, ligated with T4 ligase, the ligated product was recovered with a DNA recovery kit (Omega, D6492-02), and transformed into competent E.coli SS320 (Lucigen, MC1061F) by an electrotransfer instrument (Bio-Rad, MicroPulser), and spread on 2-YT (C10631F) containing ampicillin and tetracycline + /K + 2-YT) solid plate, amplifying SS320 bacteria of correctly transformed antibody plasmid, and finally constructing a library (hereinafter referred to as mouse immune library) containing Fab fragment antibody sequences.
3.2 phage display antibody Gene library screening
3.2.1 screening of phage display antibody Gene library by the cellular method
hCRDN18.2-HEK 293T cells or hCRDN18.1-HEK 293 cells were cultured in T25 culture flasks. When the cell growth density was close to 90%, the culture supernatant was removed and rinsed once with PBS (source culture, B310KJ), then fixed for 1 hour by adding 5mL of 4% paraformaldehyde (raw culture, E672002-0500), and finally rinsed twice with PBS as a screening material. In the screening process, the corresponding phage of the mouse immune library is firstly incubated with the fixed hLDN 18.1-HEK293 cells for 1 hour at room temperature, and then the adsorbed supernatant phage is sucked up and incubated with the fixed hLDN 18.2-HEK293T cells for 2 hours. After rinsing twice with 1 × PBS, 3mL of glycine-HCl (pH 2.0) was added and gently mixed for 10 minutes to elute phages specifically binding to human CLDN18.2, and then the eluted supernatant was infected with SS320 cells (Lucigen, 60512-1) at log phase, left to stand for 30 minutes, then cultured at 220rpm for 1 hour, and then helper phages were added by VSCM13, left to stand for 30 minutes, continued to be cultured at 220rpm for 1 hour, centrifuged and replaced to C + /K + 2-YT medium, the resulting phage was used for a second round of selection. This was repeated, and the library was evaluated by sequence analysis of 10 randomly selected clones in each round, and after 3 rounds of screening, the sequence enrichment in the library was significant.
3.2.2 selection of monoclonal
hLDN 18.2-HEK293T cells and hLDN 18.1-HEK293 cells were separately centrifuged at a low speed of 500g to remove the supernatant, the cells were washed with FACS buffer, and then phage blocked with 10% FBS (Gibco, 15140-: 50 murine monoclonal antibody (Sino Biological, 11973-MM05T) was added to M13 and incubated at 4 ℃ for 1 hour. Then rinsed twice with FACS buffer, an APC-labeled anti-mouse Fc secondary antibody (Jackson, 115136071) was added, incubated at 4 ℃ for 1 hour, rinsed twice with FACS buffer, and finally detected by flow cytometry (Beckman, CytoFLEX, AOO-1-1102). As a result, as shown in fig. 1, the phages of clone No. 188 and clone No. 262 (surface display corresponding Fab fragments) from the mouse immune pool bound to hcldn18.1-HEK293 cells and hcldn18.2-HEK293T, respectively, and clone No. 262 had excellent properties of high recognition of human CLDN18.2 and low recognition of human CLDN 18.1.
Candidate antibodies were named by clone number and the complementarity determining regions sequences of the heavy and light chains of the antibodies were determined in such a way that the CDRs were defined by AbM. The CDR amino acid sequence of candidate antibody 262 is shown in Table 1, and the amino acid sequences of variable regions VH and VL are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
TABLE 1 CDR amino acid sequences of exemplary antibodies (candidate antibodies) of the invention
Figure BDA0002981156570000201
Example 4 construction, expression and purification of chimeric antibodies
4.1 plasmid construction
The VH in the Fab sequence specifically binding to human CLDN18.2 obtained by screening is fused with the constant region (SEQ ID NO:11) of human IgG1 to construct the heavy chain (SEQ ID NO:12) of the human-mouse chimeric antibody 262, the VL in the Fab sequence specifically binding to human CLDN18.2 obtained by screening is fused with the human Kappa constant region (SEQ ID NO:13) to construct the light chain (SEQ ID NO:14) of the human-mouse chimeric antibody 262, and the constructed heavy chain and light chain are inserted into the expression plasmid pcDNA3.4.
4.2 antibody expression purification
Expression of the antibody was performed using the ExpicHO transient expression system (Gibco, A29133) and the transfection kit (Gibco, A29129). The specific method comprises the following steps: the ExpCHO cells are passaged the day before transfection, 25 mu g of the constructed plasmid and a transfection reagent are mixed in a 25mL system and then dripped into 25mL of the ExpCHO cells, and after the mixture is fully mixed, the mixture is expressed for 18 to 22 hours in a cell culture box at 37 ℃. Subsequently, a feed medium (Gibco, A29100-01) was added to the above transfection mixture and the culture was continued in a cell culture chamber at 32 ℃. On day 5 post-transfection, a second feed was added and the cells placed in a 32 ℃ cell incubator for further 10-12 days. Then, the cell suspension after expression was centrifuged at high speed, and the supernatant was collected, filtered through a 0.22 μm filter and purified by protein A/G affinity column chromatography. And after purification, eluting the target protein by using 100mM glycinate (pH is 3.0), concentrating, replacing, subpackaging, and identifying by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and detecting the SEC purity to obtain the anti-CLDN 18.2 human-mouse chimeric antibody 262 with the SEC purity higher than 94%.
Example 5 cell-specific binding assay for human murine chimeric antibody 262
hCDN18.2-HEK 293T cells, HEK293T cells, HEK293 cells and hCDN18.1-HEK 293 cells in good growth state were digested with Trypsin (Gibco, 25200-072) containing 0.25% EDTA, and 1X 10 cells were collected 5 The cells were incubated with 3-fold gradient dilutions of human murine chimeric antibody 262 for 1 hour, with an irrelevant antibody of the hIgG1 isotype as control. The cells were rinsed twice with FACS buffer and incubated with 0.5. mu.g of a PE-labeled goat anti-human IgG Fc secondary antibody (Abcam, ab98596) for 1 hour at 4 ℃. Thereafter, FACS buffers were washed three times and binding of the candidate antibody to the cells was detected by flow cytometry (Beckman, CytoFLEX, AOO-1-1102). The results of the flow assay were as follows: FIG. 2a results show that human murine chimeric antibody 262 has superior binding to hCDN18.2-HEK 293T cells as compared to the control antibody IMAB362(Zolbetuximab), where the EC of human murine chimeric antibody 262 is higher 50 EC of IMAB362 ═ 3.9nM 50 6.4 nM; the results in FIG. 2b show that the binding capacity of the human murine chimeric antibody 262 to hCDN18.2-KATOIII cells is significantly better than that of the control antibody IMAB362, wherein the EC of 262 is 50 4.7nM EC of IMAB362 50 9.8 nM; the results in FIG. 2c show that the binding of the human murine chimeric antibody 262 to hLDN 18.1-HEK293 cells is comparable to the control antibody IMAB 362.
Example 6 antibody humanization engineering
The heavy chain and light chain V regions of the chimeric human-mouse antibody are subjected to amino acid point mutation to make the chimeric human-mouse antibody closer to human Germinine. Wherein the candidate antibody is human murine chimeric antibody 262, and the partial candidate antibodies after modification are 262-H1L1, 262-H1L2, 262-H2L1 and 262-H2L 2. Wherein, the amino acid sequences of the heavy chain variable region 262-H1 and 262-H2 of the humanized antibody are respectively shown as SEQ ID NO. 15 and SEQ ID NO. 16, and the amino acid sequences of the light chain variable region 262-L1 and 262-L2 of the humanized antibody are respectively shown as SEQ ID NO. 17 and SEQ ID NO. 18.
Example 7 cell-specific binding assay for humanized antibodies
hCRDNN 18.2-HEK293T cells, hCRDNN 18.2-KATOIII cells or hCRDNN 18.1-HEK293 cells in good culture were incubated with human murine chimeric antibody 262, humanized antibodies 262-H1L1, 262-H1L2, 262-H2L1 and 262-H2L2 (three-fold gradient dilution) at 4 ℃ for 1 hour. After rinsing twice with FACS buffer, 0.5. mu.g of goat anti-human IgG Fc antibody (Abcam, ab98596) labeled with PE was added, and the mixture was incubated at 4 ℃ for 1 hour, and after rinsing twice with FACS buffer, the mixture was examined by flow cytometry.
The flow detection results are as follows: the results in FIG. 3a show that 262-H1L1, 262-H2L1, 262-H1L2 and 262-H2L2 bind hCRDN 18.2-HEK293T cells with comparable capacity to the control antibody IMAB 362; the results in FIG. 3b show that the binding capacity of 262-H1L2 and 262-H2L2 to hLDN 18.2-KATOIII cells is significantly better than that of the control antibody IMAB362, wherein the EC of 262-H1L2 50 EC of 4.1nM, 262-H2L2 50 EC of IMAB362 ═ 3.9nM 50 9.8 nM; the results in FIG. 3c show that humanized antibody 262-H2L2 binds to hCLDNN 18.1-HEK293 cells minimally and comparably to the control antibody IMAB 362.
Example 8 CDC Effect of human murine chimeric antibody 262 and humanized antibodies thereof
Digesting hLDN18.2-HEK 293T or hLDN18.2-KATOIII cells in good culture state by Trypsin, and collecting 5 × 10 4 The cells were mixed with 5-fold diluted rabbit serum, 50. mu.L of human murine chimeric antibody 262, humanized antibody 262-H1L2, humanized antibody 262-H2L2 or control antibody IMAB362 (3-fold gradient dilution) were added, respectively, and incubated at 37 ℃ for 3 hours. Then adding 30 μ L of MTS reagent (Promega, G3580) to candidate antibody or control antibody IMAB362, mixing well, and standing at 37 deg.C and 5% CO 2 The incubation chamber of (2) was incubated for 4 hours, during which time the color change of the medium was observed, and the OD492 value was measured by a microplate reader. Wherein TritonX-100 with a concentration of 10% is added into target cells as a control for total lysis, only the target cells are added as a blank control, and rabbit complement plus the target cells are added as a background negative control.
The cell killing rate was calculated as follows: cell killing (%) - (candidate antibody well OD value-background well OD value)/(total lysis well OD value-blank well OD value) × 100%. The MTS method has the following detection results:
as shown in FIG. 4a, CDC effect of human murine chimeric antibody 262 on hCDN18.2-HEK 293T cells was superior to that of the control antibody IMAB362, wherein the EC of human murine chimeric antibody 262 50 0.28. mu.g/mL, EC of IMAB362 50 0.62 μ g/mL; as shown in FIG. 4b, CDC killing effect of human murine chimeric antibody 262 on hCLDNN 18.2-KATOIII cells was significantly better than that of the control antibody IMAB362, wherein EC of human murine chimeric antibody 262 was 50 1.1. mu.g/mL, EC of IMAB362 50 ＝25.3μg/mL。
As shown in FIG. 5a, CDC effect of humanized antibody 262-H1L2 on hCLDN18.2-HEK293T cells was superior to that of control antibody IMAB362, in which the EC of humanized antibody 262-H1L2 was 50 EC of IMAB362 ═ 0.15. mu.g/mL 50 0.72 μ g/mL; as shown in FIG. 5b, CDC effect of humanized antibody 262-H1L2 on hCLDNN 18.2-KATOIII cells was significantly better than that of the control antibody IMAB362, in which the EC of humanized antibody 262-H1L2 50 0.86. mu.g/mL, EC of IMAB362 50 ＝18.1μg/mL。
As shown in FIG. 6a, the CDC effect of humanized antibody 262-H2L2 on hCLDN18.2-HEK293T cells was superior to that of the control antibody IMAB362, in which the EC of humanized antibody 262-H2L2 50 0.13. mu.g/mL, EC of IMAB362 50 1.04 μ g/mL; as shown in FIG. 6b, CDC effect of humanized antibody 262-H2L2 on hCLDNN 18.2-KATOIII cells was significantly better than that of the control antibody IMAB362, in which the EC of humanized antibody 262-H2L2 50 0.47. mu.g/mL, EC of IMAB362 50 ＝16.3μg/mL。
Example 9 ADCC Effect of human murine chimeric antibody 262 and humanized antibody thereof
50 μ L of 2X 10 density per well in 96-well cell culture plates 5 Each/mL of hLDN 18.2-KATOIII cells was cultured overnight (16-20 hours) in a 37 ℃ incubator. Adding 50 μ L of human-mouse chimeric antibody 262, humanized antibody 262-H1L2, humanized antibody 262-H2L2 or control antibody IMAB362, mixing, incubating at 37 deg.C for 20 min, and adding resuscitated antibody5X 10 of 5 Human PBMC cells (effector cell/target cell ratio 50: 1) of each well were incubated in an incubator at 37 ℃ for 4 hours, then the supernatant was centrifuged at 300g, then LDH detection reagent (Takara, MK401) was added to the supernatant to react for 1 hour, and finally OD at 492nm was measured by a microplate reader (molecular devices, SpectraMax 190) and the results were analyzed. Wherein, 10% TritonX-100 plus target cells are used as a control of total lysis, only target cells are used as a blank negative control, and PBMC plus target cells are used as a background negative control.
The cell killing rate was calculated as follows: the killing rate (%) — x 100% (candidate antibody well OD value-background well OD value)/(full lysis well OD value-blank well OD value). The ADCC detection result is as follows:
as shown in FIG. 7, ADCC killing effect of human murine chimeric antibody 262 on hCLDN18.2-KATOIII cells was significantly better than that of the control antibody IMAB362, wherein EC of human murine chimeric antibody 262 was 50 EC of IMAB362 0.09nM 50 ＝0.57nM。
As shown in FIG. 8, the ADCC effect of humanized antibody 262-H1L2 on hCRND18.2-KATOIII cells was superior to that of the control antibody IMAB362, in which the EC of humanized antibody 262-H1L2 was better 50 0.78nM, EC of IMAB362 50 ＝0.98nM。
As shown in FIG. 9, the ADCC effect of humanized antibody 262-H2L2 on hCDN18.2-KATOIII cells was significantly better than that of the control antibody IMAB362, in which the EC of humanized antibody 262-H2L2 was 50 0.12nM, EC of IMAB362 50 ＝0.74nM。
Example 10 antibody engineering of humanized antibody 262-H2L2
In order to promote specific binding of the antibody molecule to human CLDN18.2 and further reduce non-specific binding to human CLDN18.1, this example was antibody engineered to humanized antibody 262-H2L 2.
Performing single-point or continuous two-point mutation on the CDR defined by the AbM to construct an affinity maturation phage display library, and screening molecules after affinity maturation by using a phage display technology, wherein the screening method refers to example 3.2. Through 3 rounds of affinity maturation modification, modified antibodies are obtained, which are 262-H2L2-7, 262-H2L2-11, 262-H2L2-23, 262-H2L2-24, 262-H2L2-53, 262-H2L2-2, 262-H2L2-9, 262-H2L2-14, 262-H2L2-19 and 262-H2L2-20 respectively. The complementarity determining region sequences of the heavy and light chains of an antibody are determined in the manner in which AbM defines CDRs. The amino acid sequences of each candidate engineered antibody are shown in tables 2 and 3.
TABLE 2 CDR amino acid sequences of exemplary engineered antibodies of the invention
Figure BDA0002981156570000241
TABLE 3 variable region amino acid sequences of exemplary engineered antibodies of the invention
Figure BDA0002981156570000242
Figure BDA0002981156570000251
Example 11 binding of engineered antibodies to cells overexpressing human CLDN18.2 or overexpressing human CLDN18.1
hCLDNN 18.2-HEK293 cells or hCLDNN 18.1-HEK293 cells in a good culture state are taken and respectively incubated with 262-H2L2 and partially-modified antibodies 262-H2L2-7, 262-H2L2-11, 262-H2L2-23, 262-H2L2-24 and 262-H2L2-53 (which are all diluted by 3 times of gradient) at 4 ℃ for 1 hour. After rinsing twice with FACS buffer, 0.5. mu.g of goat anti-human IgG Fc antibody labeled with PE (Abcam, ab98596) was added, and the mixture was incubated at 4 ℃ for 1 hour, and after rinsing twice with FACS buffer, the mixture was examined by flow cytometry.
The results of the flow assay are as follows: FIG. 10a shows that the binding capacity of the engineered antibodies 262-H2L2-7, 262-H2L2-11, 262-H2L2-23, 262-H2L2-24, 262-H2L2-53 to hLDN 18.2-HEK293 cells is equivalent to that of the humanized antibody 262-H2L 2; the results in FIG. 10b show that the modified antibodies 262-H2L2-7, 262-H2L2-11, 262-H2L2-23, 262-H2L2-24 and 262-H2L2-53 have no significant binding with hLDN 18.1-HEK293 cells, and the effect is equivalent to that of unrelated antibodies.
Example 12 non-specific binding of engineered antibodies to CHO-K cells
CHO-K cells in good culture, the humanized antibody 262-H2L2 and the modified antibody (both antibodies were diluted in a 3-fold gradient) were incubated at 4 ℃ for 1 hour. After rinsing twice with FACS buffer, 0.5. mu.g of goat anti-human IgG Fc antibody (Abcam, ab98596) labeled with PE was added, and the mixture was incubated at 4 ℃ for 1 hour, and after rinsing twice with FACS buffer, the mixture was examined by flow cytometry.
The results of the flow assay were as follows: FIG. 11 shows that the engineered antibodies 262-H2L2-7, 262-H2L2-11, 262-H2L2-23, 262-H2L2-24, 262-H2L2-53 bind significantly less strongly to CHO-K cells than the control antibody IMAB362, and thus the specificity of the engineered antibody is better.
Example 13 CDC Effect of engineered antibodies
In this example, the CDC cell killing effect of the candidate antibody was measured by MTS method, and the specific method was performed in reference to example 8, and the antibody concentrations are shown in FIGS. 12a to 12 e.
CDC assay results were as follows: as shown in FIG. 12a, the CDC killing effect of the engineered antibody 262-H2L2-7 on hLDN 18.2-HEK293 cells is significantly better than that of the control antibody IMAB362, wherein the EC of the engineered antibody 262-H2L2-7 50 0.4113nM EC of IMAB362 50 2.042 nM; as shown in FIG. 12b, the CDC killing effect of the engineered antibody 262-H2L2-11 on hCLDN18.2-HEK293 cells was superior to that of the control antibody IMAB362, wherein the EC of the engineered antibody 262-H2L2-11 50 0.7569nM EC of IMAB362 50 1.841 nM; as shown in FIG. 12c, the CDC killing effect of the engineered antibody 262-H2L2-23 on hLDN 18.2-HEK293 cells was significantly better than that of the control antibody IMAB362, wherein the EC of the engineered antibody 262-H2L2-23 50 0.6943nM EC of IMAB362 50 2.428 nM; as shown in FIG. 12d, the CDC killing effect of the engineered antibody 262-H2L2-24 on hCLDN18.2-HEK293 cells was similar to that of the humanized antibody 262-H2L2, but significantly better than that of the control antibody IMAB362, wherein the EC of the engineered antibody 262-H2L2-24 50 EC of humanized antibody 262-H2L2 at 0.4431nM 50 0.3432nM EC of IMAB362 50 1.748 nM; as shown in FIG. 12e, the CDC killing effect of the engineered antibody 262-H2L2-53 on hCLDNN 18.2-HEK293 cells was similar to that of the humanized antibody 262-H2L2, but significantly better than that of the control antibody IMAB362, wherein the EC of the engineered antibody 262-H2L2-53 50 EC of humanized antibody 262-H2L2 ═ 0.3995nM 50 0.3530nM EC of IMAB362 50 ＝2.054nM。
Example 14 ADCC Effect of the engineered antibody
After the Fc end of the antibody targeting CLDN18.2 is combined with CD16a (F158) or CD16a (V158) and the Fab end is combined with hCDN 18.2-HEK293, the expression of NF-AT protein in Jurkat cells is activated, the expression of luciferase downstream of the Jurkat cells is triggered by the combination of the NF-AT and a reaction element of the NF-AT, and the ADCC activity of the antibody is evaluated by obtaining a fluorescence reading curve with antibody concentration dependence by stimulating the antibody with different concentration gradients.
50 μ L of a 4X 10 density solution was added to each well of a 96-well cell culture plate 5 hCLDNN 18.2-HEK293 cells and 4X 10 cells per mL 6 CD16a (F158) -NF-AT-jurkat cells/mL, cells 1:1 mixed and added to a 96-well white-edged cell-penetrating cell culture plate, and incubated overnight (16-20 hours) in an incubator AT 37 ℃. 50 μ L of gradient diluted engineered antibodies 262-H2L2-7, 262-H2L2-23, humanized antibody 262-H2L2, control antibody IMAB362, control antibody hu8E5-2I (prepared by expression and purification of the sequence information of heavy chain SEQ ID NO:63 and light chain SEQ ID NO:65 disclosed in patent WO2018006882A 1), control antibody ASK589-M5 (prepared by expression and purification of the sequence information of heavy chain SEQ ID NO:257 and light chain SEQ ID NO:260 disclosed in patent US 20200040101A 1) or control antibody 190h 1-11 (prepared by expression and purification of the sequence information of heavy chain SEQ ID NO:44 and light chain SEQ ID NO:41 disclosed in patent WO2020200196A 1) were added, and the incubator was incubated at 37 ℃ for 6H. mu.L of Bright-Life (vazyme, cat # DD1204-03) was added to each well, and incubated for 10min in the absence of light, and the fluorescent signal was detected.
The ADCC detection result is as follows: as shown in FIG. 13a, the ADCC killing effect of the engineered antibody 262-H2L2-7 on hLDN 18.2-HEK293 cells is significantly better than that of the control antibodies IMAB362 and hu8E5-2I, wherein the antibody 262-H2L2-7 is obtained after the engineeringEC 50 EC of control antibody IMAB362 ═ 0.031 μ g/mL 50 EC of control antibody hu8E5-2I ═ 0.281 μ g/mL 50 0.064 μ g/mL; as shown in FIG. 13b, the ADCC killing effect of the modified antibody 262-H2L2-7 on hCLDN18.2-HEK293 cells is obviously better than that of the control antibodies ASK589-M5 and H1901-11, wherein the EC of the modified antibody 262-H2L2-7 50 EC of control antibody ASK589-M5 ═ 0.033 μ g/mL 50 EC of control antibody h1901-11 (0.091. mu.g/mL) 50 0.079 μ g/mL; as shown in FIG. 13c, the ADCC killing effect of the engineered antibody 262-H2L2-23 on hLDN 18.2-HEK293 cells was significantly better than that of the control antibodies IMAB362 and hu8E5-2I, wherein the EC of the engineered antibody 262-H2L2-23 50 EC of control antibody IMAB362 ═ 0.047 μ g/mL 50 EC of control antibody hu8E5-2I of 0.339 μ g/mL 50 0.104 μ g/mL; as shown in FIG. 13d, the ADCC killing effect of the engineered antibody 262-H2L2-23 on hCDN18.2-HEK 293 was significantly better than that of the control antibodies ASK589-M5 and H1901-11, wherein the EC of the engineered antibody 262-H2L2-23 50 EC of control antibody ASK589-M5 ═ 0.046 μ g/mL 50 EC of control antibody h1901-11 ═ 0.105. mu.g/mL 50 ＝0.085μg/mL。
Example 15 in vivo tumor inhibition assay
Female SCID mice (24-26g) with the age of 6-8 weeks are used and are raised in an independent ventilation box with constant temperature and humidity, the temperature of the raising room is 21-24 ℃, and the humidity is 30-53%. Will be 1 × 10 7 An injection of hLDN 18.2-HEK293T cells was injected subcutaneously into the right axilla to eliminate mice with large differences in tumor volume. Random groupings were made by tumor volume (8 mice per group): the antibody treatment groups comprise a PBS treatment group, an IMAB362 antibody treatment group and an engineered antibody treatment group, wherein the engineered antibodies are selected from 262-H2L2-2, 262-H2L2-9, 262-H2L2-14, 262-H2L2-19, 262-H2L2-20, 262-H2L2-7 and 262-H2L2-23 respectively. The administration treatment was started 14 days after the cell inoculation, and the administration treatment was performed 2 times per week by alternating intravenous injection and intraperitoneal injection, respectively. Tumor length (L) and width (W) were observed and recorded over time, and tumor growth volume (V) was calculated by: v ═ lxw 2 /2。
The result shows that the modified antibody has the effect of inhibiting tumors. As shown in FIG. 14, at the same dose of 10mg/kg, IMAB362, 262-H2L2-2 and 262-H2L2-9 had better tumor suppression effect than the control antibodies IMAB362, 262-H2L2-2 and 262-H2L 2-9.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Sanyou biomedical (Shanghai) Co., Ltd
<120> an antibody targeting CLDN18.2 or an antigen-binding fragment thereof and use thereof
<130> P20017633C
<160> 46
<170> PatentIn version 3.5
<210> 1
<211> 261
<212> PRT
<213> Homo sapiens
<400> 1
Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu
1 5 10 15
Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val
260
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Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
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Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
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Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
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Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
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Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
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Lys His Asp Tyr Val
260
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Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly
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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser
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Thr Met Ser Trp Val Arg Gln Thr Pro Ala Lys Arg Leu Glu Trp Val
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Ala Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser Tyr Pro Asp Ser Val
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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr
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Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
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Ala Arg Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asp
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Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
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Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
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Gln Ser Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
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Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
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Lys Leu Leu Ile Tyr Gly Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Asp Asp Ile Gly Met Tyr Phe Cys Gln Gln Ser Arg
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Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Gly Phe Thr Phe Ser Ser Ser Thr Met Ser
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Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser
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Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asp Tyr
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Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln
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Gly Ala Ser Asn Val Glu Ser
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Gln Gln Ser Arg Lys Val Pro Trp Thr
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
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Ser Thr Ser Gly Gly 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 Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
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Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
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Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
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Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
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Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
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Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
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Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
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Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
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Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
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Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
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Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
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Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
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Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly
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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser
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Thr Met Ser Trp Val Arg Gln Thr Pro Ala Lys Arg Leu Glu Trp Val
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Ala Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser Tyr Pro Asp Ser Val
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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr
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Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
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Ala Arg Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asp
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Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys
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Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
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Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
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Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
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Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
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Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
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Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro
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Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
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Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
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Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
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Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
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Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
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Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
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Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
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Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
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Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
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Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
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Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys
450
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<213> Homo sapiens
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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
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Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
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Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 14
<211> 218
<212> PRT
<213> Artificial Sequence
<220>
<223> light chain of human murine chimeric antibody 262
<400> 14
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
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Gln Ser Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Gly Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Asp Asp Ile Gly Met Tyr Phe Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 15
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> heavy chain variable region 262-H1 of humanized antibody
<400> 15
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 Phe Thr Phe Ser Ser Ser
20 25 30
Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Arg Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 16
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> heavy chain variable region 262-H2 of humanized antibody
<400> 16
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 Phe Thr Phe Ser Ser Ser
20 25 30
Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Arg Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 17
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> light chain variable region 262-L1 of humanized antibody
<400> 17
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Gly Ala Ser Asn Val Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 18
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> light chain variable region 262-L2 of humanized antibody
<400> 18
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 Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 19
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-7 VL
<400> 19
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 Arg Ala Ser Glu Ser Val Ser Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Trp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 20
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-23 VL
<400> 20
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 Arg Ala Ser Glu Ser Val Ser Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Trp Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 21
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-24 VL
<400> 21
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 Arg Ala Ser Glu Ser Val Ser Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Glu Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Trp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 22
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-53 VL
<400> 22
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 Arg Ala Ser Glu Ser Val Ser Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Trp Val Pro Leu Val Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 23
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-7/11/23/24/53/2 L-CDR1
<400> 23
Arg Ala Ser Glu Ser Val Ser Tyr Tyr Gly Thr Ser Leu Met Gln
1 5 10 15
<210> 24
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-24 L-CDR2
<400> 24
Ala Ala Ser Glu Leu Gln Ser
1 5
<210> 25
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-7/11/24 L-CDR3
<400> 25
Gln Gln Ser Arg Trp Val Pro Trp Thr
1 5
<210> 26
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-23 L-CDR3
<400> 26
Gln Gln Ser Arg Trp Val Pro Leu Thr
1 5
<210> 27
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-53 L-CDR3
<400> 27
Gln Gln Ser Arg Trp Val Pro Leu Val
1 5
<210> 28
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-7 VH
<400> 28
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 Phe Thr Phe Ser Ser Ser
20 25 30
Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Arg Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asn
100 105 110
Leu Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 29
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> SEQ ID NO:29 262-H2L2-11 VH
<400> 29
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 Arg Thr Phe Ser Ser Ser
20 25 30
Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Arg Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asn
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 30
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-23/24/53/2 VH
<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 Ala Ser Gly Phe Thr Phe Ser Ser Ser
20 25 30
Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Arg Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asn
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 31
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-11 H-CDR1
<400> 31
Gly Arg Thr Phe Ser Ser Ser Thr Met Ser
1 5 10
<210> 32
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-7 H-CDR3
<400> 32
Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asn Leu
1 5 10 15
<210> 33
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-11/23/24/53 H-CDR3
<400> 33
Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Asn Tyr
1 5 10 15
<210> 34
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-2 VL
<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 Arg Ala Ser Glu Ser Val Ser Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Phe Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 35
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-9 VL
<400> 35
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 Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Arg Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 36
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-14 VL
<400> 36
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 Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Gly Thr Arg Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 37
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-19 VL
<400> 37
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 Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Arg Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 38
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-20 VL
<400> 38
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 Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95
Phe Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 39
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-9 L-CDR1
<400> 39
Arg Ala Ser Glu Ser Val Glu Tyr Tyr Arg Thr Ser Leu Met Gln
1 5 10 15
<210> 40
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-14 L-CDR1
<400> 40
Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Arg Leu Met Gln
1 5 10 15
<210> 41
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-19 L-CDR2
<400> 41
Ala Ala Ser Asn Arg Gln Ser
1 5
<210> 42
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-2 L-CDR3
<400> 42
Gln Gln Ser Arg Phe Val Pro Trp Thr
1 5
<210> 43
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-9 L-CDR3
<400> 43
Gln Gln Ser Arg Lys Val Pro Trp Ser
1 5
<210> 44
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-9 VH
<400> 44
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 Phe Thr Phe Ser Ser Ser
20 25 30
Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Gly Gly Gly Gly Gly Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Arg Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Pro
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 45
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> 262-H2L2-9 H-CDR3
<400> 45
Gln Gly Gly Ser Phe Tyr Tyr Tyr Asp Gly Ser Gly Phe Pro Tyr
1 5 10 15
<210> 46
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> 262- H2L2-23 L-CDR2
<400> 46
Ala Ala Ser Asn Leu Gln Ser
1 5

Claims (24)

1. An antibody or antigen-binding fragment thereof targeting CLDN18.2 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3,
the HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 5 or a variant 1 thereof, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 6, the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 7 or a variant 2 thereof, the LCDR1 comprises an amino acid sequence shown as SEQ ID NO. 8 or a variant 3 thereof, the LCDR2 comprises an amino acid sequence shown as SEQ ID NO. 9 or SEQ ID NO. 46 or a variant 4 thereof, and the LCDR3 comprises an amino acid sequence shown as SEQ ID NO. 10 or a variant 5 thereof;
the variant is a1, 2 or 3 amino acid substitution, deletion or addition based on the original sequence, and an antibody or antigen-binding fragment thereof comprising the variant retains at least the binding ability to CLDN 18.2.
2. The antibody or antigen-binding fragment thereof of claim 1,
the mutation of the variant 1 at least occurs at the 2 nd position of the amino acid sequence shown in SEQ ID NO. 5;
the mutation of the variant 2 at least occurs at the 14 th and/or 15 th position of the amino acid sequence shown in SEQ ID NO. 7;
the mutation of the variant 3 occurs at least at one or more of positions 7, 10 and 12 of the amino acid sequence shown in SEQ ID NO. 8;
the mutation of the variant 4 at least occurs at the 4 th and/or 5 th position of the amino acid sequence shown in SEQ ID NO. 46;
the mutation of the variant 5 occurs at least at one or more of positions 5, 8 and 9 of the amino acid sequence shown in SEQ ID NO. 10; wherein:
the preferred mutation at position 2 in variant 1 is R;
in the variant 2, the 14 th position is preferably mutated into N or P, and the 15 th position is preferably mutated into L;
in the variant 3, the 7 th preferred mutation is S, the 10 th preferred mutation is R, and the 12 th preferred mutation is R;
in the variant 4, the 4 th preferred mutation is E, and the 5 th preferred mutation is R;
in the variant 5, the 5 th site is preferably mutated into W or F, the 8 th site is preferably mutated into L, and the 9 th site is preferably mutated into V or S.
3. The antibody or antigen-binding fragment thereof of claim 2, wherein the amino acid sequence of variant 1 is set forth in SEQ ID No. 31;
and/or the amino acid sequence of the variant 2 is shown as SEQ ID NO:32, 33 or 45;
and/or the amino acid sequence of the variant 3 is shown as SEQ ID NO. 23, 39 or 40;
and/or the amino acid sequence of the variant 4 is shown as SEQ ID NO. 24 or 41;
and/or the amino acid sequence of the variant 5 is shown as SEQ ID NO 25, 26, 27, 42 or 43.
4. The antibody or antigen-binding fragment thereof of claim 3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO. 5, the amino acid sequence of HCDR2 is set forth in SEQ ID NO. 6, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO. 7;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 45;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 32;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 31, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33;
preferably, the framework region of the heavy chain variable region is a human or murine framework region;
more preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO 3, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30 or SEQ ID NO 44.
5. The antibody or antigen-binding fragment thereof of claim 3 or 4, wherein the amino acid sequence of LCDR1 is set forth in SEQ ID NO. 8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO. 9, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO. 10;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 25;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 26;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 24, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 25;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 27;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 42;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 39, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 43;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 40, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 10;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 41, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 10;
or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 42;
preferably, the framework region of the light chain variable region is a human or murine framework region;
more preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37 or SEQ ID NO. 38.
6. The antibody or antigen-binding fragment thereof of claim 5, wherein the amino acid sequence of HCDR1 is set forth as SEQ ID NO. 5, the amino acid sequence of HCDR2 is set forth as SEQ ID NO. 6, the amino acid sequence of HCDR3 is set forth as SEQ ID NO. 7, the amino acid sequence of LCDR1 is set forth as SEQ ID NO. 8, the amino acid sequence of LCDR2 is set forth as SEQ ID NO. 9, and the amino acid sequence of LCDR3 is set forth as SEQ ID NO. 10;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 7, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 40, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 10;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 7, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 41, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 10;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 7, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 42;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 32, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 25;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 25;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 26;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 24, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 25;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 27;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 33, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 42;
or the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 6, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 45, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 39, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 46, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 43.
7. The antibody or antigen-binding fragment thereof of claim 6, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 3 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 4;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17;
or, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 28, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 19;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 29, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 19;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 20;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 21;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 22;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 34;
or, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 44, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 35;
or, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 36;
or, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 37;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 38.
8. The antibody or antigen-binding fragment thereof of claim 7, wherein the antibody further comprises an antibody heavy chain constant region and an antibody light chain constant region; preferably, the heavy chain constant region is derived from a heavy chain of a human antibody or a variant thereof and the light chain constant region is derived from a kappa chain or a lambda chain of a human antibody or a variant thereof.
9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antibody is a full-length antibody, Fab ', F (ab') 2 Fv, bispecific or multispecific antibodies, or monoclonal or polyclonal antibodies made from such antibodies; the Fv is preferably an scFv.
10. An isolated nucleic acid encoding the antibody having the target CLDN18.2 of any one of claims 1 to 9 or an antigen-binding fragment thereof.
11. A recombinant expression vector comprising the isolated nucleic acid of claim 10; preferably, the recombinant expression vector is a plasmid, cosmid, phage, or viral vector, preferably a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
12. A transformant comprising the recombinant expression vector of claim 11 in a host cell; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the host cell is selected from the group consisting of a yeast cell, a mammalian cell, or other cell suitable for the production of an antibody or antigen-binding fragment thereof; wherein the mammalian cell is, for example, 293 cell or CHO cell.
13. A chimeric antigen receptor comprising the antibody targeting CLDN18.2 or an antigen-binding fragment thereof of any one of claims 1-9.
14. A genetically modified cell comprising the chimeric antigen receptor of claim 13; preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably immune cells such as T cells, or NK cells.
15. A method for preparing an antibody or antigen-binding fragment thereof targeting CLDN18.2, comprising culturing the transformant of claim 12, and obtaining the antibody or antigen-binding fragment thereof targeting CLDN18.2 from the culture.
16. An antibody drug conjugate comprising a cytotoxic agent and the antibody targeting CLDN18.2 or an antigen-binding fragment thereof of any one of claims 1-9; preferably, the cytotoxic agent is MMAF or MMAE.
17. A pharmaceutical composition comprising an antibody having the target CLDN18.2 or an antigen-binding fragment thereof of any one of claims 1-9 and/or an antibody drug conjugate of claim 16, and a pharmaceutically acceptable carrier;
preferably, the pharmaceutical composition further comprises one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.
18. Use of an antibody or antigen-binding fragment thereof targeting CLDN18.2 of any one of claims 1 to 9, an antibody drug conjugate of claim 16 and/or a pharmaceutical composition of claim 17 for the preparation of a medicament for the diagnosis, prevention and/or treatment of a tumor; preferably, the tumor is a CLDN18.2 positive tumor; more preferably, the tumor is gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and/or leukemia.
19. A kit comprising an antibody having a CLDN18.2 target or an antigen-binding fragment thereof of any one of claims 1-9, a chimeric antigen receptor of claim 13, a genetically modified cell of claim 14, or an antibody drug conjugate of claim 16 or a pharmaceutical composition of claim 17;
preferably, the kit further comprises (i) a means for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
20. A kit comprising kit a and kit B, wherein:
the kit a contains an antibody having the target CLDN18.2 or an antigen-binding fragment thereof of any one of claims 1 to 9, a chimeric antigen receptor of claim 13, a genetically modified cell of claim 14, an antibody drug conjugate of claim 16, and/or a pharmaceutical composition of claim 17;
the kit B contains the other anti-tumor antibody or a pharmaceutical composition comprising the other anti-tumor antibody and/or one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.
21. A method of diagnosing, treating and/or preventing a CLDN 18.2-mediated disease or disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof targeting CLDN18.2 of any one of claims 1-9, a chimeric antigen receptor of claim 13, an antibody drug conjugate of claim 16 or a pharmaceutical composition of claim 17, or treating a patient in need thereof using the kit of claim 20.
22. The method of claim 21, wherein the disease or disorder is a tumor, preferably a CLDN18.2 positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and/or leukemia.
23. A method of immunodetection or determination of CLDN18.2 comprising use of the antibody having a target of CLDN18.2 or antigen binding fragment thereof of any one of claims 1-9, the chimeric antigen receptor of claim 13, the antibody drug conjugate of claim 16, or the pharmaceutical composition of claim 17; preferably, the detection is a detection for non-diagnostic and/or therapeutic purposes.
24. A combination therapy comprising administering an antibody targeting CLDN18.2 or an antigen-binding fragment thereof of any one of claims 1-9, a chimeric antigen receptor of claim 13, an antibody drug conjugate of claim 16 or a pharmaceutical composition of claim 17, and a second therapeutic agent, respectively, to a patient in need thereof; the second therapeutic agent preferably comprises an additional anti-tumor antibody or a pharmaceutical composition comprising the additional anti-tumor antibody, and/or one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.
CN202110287671.0A 2021-03-17 2021-03-17 Antibody targeting CLDN18.2 or antigen binding fragment thereof and application thereof Pending CN115109154A (en)

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CN115960229A (en) * 2022-09-28 2023-04-14 华润生物医药有限公司 CLDN18.2 antibody and application thereof

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WO2020114480A1 (en) * 2018-12-07 2020-06-11 Zai Lab (Shanghai) Co., Ltd. Anti-claudin antibodies and uses thereof
KR20210110339A (en) * 2018-12-28 2021-09-07 난징 진스크립트 바이오테크 컴퍼니 리미티드 claudin18.2 binding moieties and uses thereof
KR20210109520A (en) * 2018-12-28 2021-09-06 쓰촨 케룬-바이오테크 바이오파마수티컬 컴퍼니 리미티드 Antibodies and their uses

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CN115960229A (en) * 2022-09-28 2023-04-14 华润生物医药有限公司 CLDN18.2 antibody and application thereof
CN115960229B (en) * 2022-09-28 2024-02-09 华润生物医药有限公司 CLDN18.2 antibody and application thereof

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