CN111110862A - Drug conjugate of anti-CLDN 18.2 antibody, preparation method and application thereof - Google Patents
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Abstract
The invention discloses A Drug Conjugate (ADC) of an anti-CLDN 18.2 antibody, which has a structure shown as Ab- [ (L)2)n‑L1–D]y is shown in formula I, wherein D is small molecular drug with cytotoxicity, L1And L2Separately linking the drug and the antibody; n is 0 or 1; y represents the average number of D coupled to Ab, and 0<y is less than or equal to 10; the Ab is an antibody capable of specifically binding to human CLDN18.2 comprising a light chain variable region (VL) and/or a heavy chain variable region (VH) in which the anti-CLDN 18.2 antibody is correspondingly comprisedContains at least 1 specific Complementarity Determining Region (CDR) sequence or a mutant sequence thereof, said mutation retaining or improving the binding of said antibody to CLDN 18.2. The invention also discloses a pharmaceutical composition containing the ADC and a preparation method and application of the ADC. The drug conjugate of the antibody has a large safety window and low toxic and side effects, and provides a more specific, effective and better treatment option for tumor patients.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a drug conjugate of an anti-CLDN 18.2 antibody, a preparation method and application thereof.
Background
Cancer is a great threat to human health and is one of the leading causes of death in the disease field. The development of cancer treatment in various stages of undergoing surgery, chemotherapy, targeted drugs, tumor immunotherapy, combination therapy and the like has achieved tremendous success in recent years. Among many cancer patients, therapeutic measures for cancer patients such as lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, and ovarian cancer are still highly unmet. The treatment means aiming at the tumors, comprising macromolecular targeting drugs such as new monoclonal antibodies, and the combination treatment of the monoclonal antibodies and the existing tumor immunotherapy means, comprising the immune checkpoint inhibitors PD-1 and PD-L1, provide new possibilities and choices for huge unmet clinical treatment requirements.
The cell junction Claudin (Claudin or CLDN) is expressed in human, mouse and other species, is an intercellular layer sealing associated protein, and has important functions of controlling the ion flow between cell layers, maintaining the cell polarity and transferring intercellular signals. This family of proteins has been studied as early as 1998 (Furute M, Fujita K et al. J. Cell biol.1998, 141: 1539-50; Tsukita S and Furute M, 2000.J. Cell biol.149, 13-16). CLND is a large family of proteins that play an important role in cell attachment, and is associated with cell migration, lesions, and tumor infiltration metastasis. There have been 29 more CLDN family proteins found, of which CLDN18 is one. CLDN18 has two homologous molecules, designated claudin 18.1(CLDN18.1) and claudin18.2 (CLDN18.2), respectively. The human claudin 18.1 (hLDN 18.1) and the human claudin18.2 (hLDN 18.2) are highly homologous, and the amino acid homology is as high as 92%. CLDN is expressed differently in different tissues, hLDN 18.1 is expressed in normal tissues, while hLDN 18.2 is expressed in tumor tissues and is associated with tumor formation, particularly gastric cancer (Sanada Y.et. Down-regulation of the claudin-18gene, identified with respect to gene expression data analysis, in scientific cancer with an intrinsic phenotype. J. Pathol. 2006: 208(5), 633-42). CLDN18.2 is expressed very limitedly in normal tissues, only in differentiated epithelial cells of the gastric mucosa, but is particularly highly expressed in gastric Cancer including metastatic gastric Cancer tissues (Sahin U.et al Claudin-18 plaque variant 2is a pan-Cancer target capable for therapeutic antibody delivery. clin Cancer Res.2008; 14 (23): 7642-34). It was further found that CLDN18.2 is expressed in different cancer patient tissues, including approximately 70% gastric cancer, 50% pancreatic cancer, 30% esophageal cancer, 25% lung cancer, ovarian cancer, and the like.
Therefore, CLDN18.2 has long been an ideal tumor patient marker and an anti-tumor drug development target, especially the development of anti-CLDN 18.2 antibody for tumor therapy, and meeting the requirements of patients, especially patients with gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer and the like, is one of very promising means. However, because of the specificity of the target, it is very difficult to develop therapeutic antibodies against hcldn 18.2.
The human CLDN18.2 protein is 261 amino acids in length, as disclosed in NCBI published sequence NP _001002026.1claudin-18isoform 2, where 1-23 are signal peptides. The CLDN18.2 protein is a transmembrane protein with two Extracellular regions, Extracellular region 1 (ECL1) and 23 amino acids ECL2, respectively, of about 55 amino acids behind the signal peptide. This structure is very similar to human CLDN18.1, and the ECL2 regions of human CLDN18.2 and human CLDN18.1 are identical. Therefore, the development of antibodies against a target of human CLDN18.2 protein requires the search for antibodies against the ECL1 region of human CLDN18.2 protein or for antibodies against the spatial structure of human CLDN18.2 membrane protein. This makes this aspect of the work more difficult.
Furthermore, antibodies directed against the human CLDN18.2 membrane protein exert their effects including at least induction of tumor cell apoptosis, inhibition of tumor cell growth, killing of tumor cells by effects with patient immune cells, including antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) effector cell-mediated killing. And finding antibodies that function as such is more difficult. Only IMAB362 antibody (see WO2014/146672) is currently studied for human CLDN18.2 antibody at the clinical trial stage. IMAB362 is an antibody against human CLDN18.2, is a murine chimeric antibody, and presents an immunogenic risk. And the tumor model can not be endocytosed in cytology experiments, so the treatment effect is very limited, and the tumor model is verified in the evaluation of the drug effect.
The Antibody Drug Conjugates (ADC) consist of an antibody (targeting), a joint and a small molecule drug. The antibody or the antibody fragment is connected with the cytotoxin with biological activity through a stable chemical linker compound, so that the specificity of the antibody for the combination of tumor cells or high-expression antigens and the high efficiency of the cytotoxin are fully utilized, and the toxic and side effects on normal cells are avoided. This means that the antibody drug conjugate can bind to tumor cells precisely and reduce the effect on normal cells, compared to conventional chemotherapeutic drugs. However, no ADC drugs have yet been developed clinically for the CLDN18.2 target.
Therefore, there is a lack in the art of effective drug conjugates of antibodies against human CLDN18.2 protein, in particular humanized anti-human CLDN18.2 antibodies, to reduce the potential immunogenicity in therapy, and there is also a need for drug conjugates of antibodies with better activity, including better binding activity, effector cell activity, tumor killing activity and drug efficacy, to meet the needs of patients in the field related to therapy and therapy.
Disclosure of Invention
In order to overcome the defect that the field lacks a drug conjugate of an antibody against human CLDN18.2 protein, the present invention provides a drug conjugate of an anti-CLDN 18.2 antibody, a preparation method and use thereof.
The present invention provides a drug conjugate of a CLDN18.2 antibody specifically binding to a human CLDN18.2 protein (hcldn18.2 protein), wherein the structure of the drug conjugate of the antibody is represented by formula I below:
Ab-[(L2)n-L1–D]yformula I
Wherein D is small molecular drug with cytotoxicity, L1And L2Separately linking the drug and the antibody; n is 0 or 1; y represents the average number of D coupled to Ab, and 0<y is 10, preferably 2. ltoreq. y.ltoreq.7; more preferably 3. ltoreq. y.ltoreq.6; most preferably 4.4 or 4.8;
the Ab is an antibody capable of specifically binding to human cell connexin 18.2, comprising a light chain variable region (VL) and/or a heavy chain variable region (VH) in which the anti-CLDN 18.2 antibody comprises at least 1 Complementarity Determining Region (CDR) sequence or mutated sequence thereof, respectively, selected from the group consisting of:
for the light chain variable region (VL): a VL CDR1 amino acid sequence shown as SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 22, SEQ ID NO. 23 or SEQ ID NO. 71 or SEQ ID NO. 74; a VL CDR2 amino acid sequence shown in SEQ ID NO. 13 or SEQ ID NO. 24; the amino acid sequence of VL CDR3 shown in SEQ ID NO. 14 or SEQ ID NO. 25;
for the heavy chain variable region (VH): a VH CDR1 amino acid sequence shown in SEQ ID NO. 15, SEQ ID NO. 18, SEQ ID NO. 20 or SEQ ID NO. 26; a VH CDR2 amino acid sequence shown in SEQ ID NO 16, SEQ ID NO 19, SEQ ID NO 21 or SEQ ID NO 27; the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or SEQ ID NO. 28; the mutation maintains or improves binding of the antibody to CLDN 18.2.
In the preparation of the antibody drug conjugate, the amount of the drug carried by the antibody is determined by the method of conjugation in case of site-directed conjugation, and the antibody drug conjugate may be a single product (non-mixture). In the case of site-directed random conjugation, the number of drug conjugate molecules carried by different antibodies is actually different, so that the drug conjugate of the antibody of the present invention is a mixture, and y in the formula reflects the average value of the drug conjugate carried by the antibody in the mixture. Which after numerical calculation usually appear as a non-integer positive number, e.g. 4.4 or 4.8. The average number of drug moieties per antibody in the ADC preparation from the conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assay and HPLC. Quantitative distribution of ADCs can also be determined, and in some cases, separation, purification, and characterization of homogeneous ADCs with y at a certain value from ADCs with other drug loadings can be achieved by methods such as reverse phase HPLC or electrophoresis.
In the general formula of the invention, the linker L2If present (i.e., n is 1), then L2、L1And D ofThe same amount of the antibody conjugate is Ab- [ L [ ]2-L1–D]y(ii) a If L is2L is absent (i.e. n is 0), i.e. in the case where only one linker is included, L1And D are the same in number, in which case the antibody drug conjugate would actually be Ab- [ L [ ]1–D]y. The drug conjugate of the antibody represented by the general formula of the present invention is a drug conjugate of an antibody in an ideal state, that is, the general formula considers that only the linker L is linked to1L of2Number of (if present), L to which D is attached1The number of the cells. However, it is known to those skilled in the art that in the drug conjugate of the actually synthesized antibody, there should be a linker not linked to D, so the average number of drug molecules actually conjugated to the antibody should be ≦ y, i.e., y is the theoretical maximum for the drug conjugated to the antibody.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the above antibody, wherein the small molecule drug is a cytotoxic agent selected from the group consisting of toxins, chemotherapeutic agents, antibiotics, radioisotopes, and nucleolytic enzymes.
Preferably, the small molecule drug is selected from the group consisting of: monomethyl auristatin (maytansinoid), maytansinoids, camptothecin (camptothecin), calicheamicin, doxorubicin (adriamycin), duocarmycin (duocarmycin), or combinations thereof.
More preferably, the monomethyl auristatin is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), and the maytansinoid is N2' -Deacetyl-N2' - (3-mercapto-1-oxopropyl) -maytansine (DM1), N2' -Deacetyl-N2' - (4-mercapto-1-oxopentyl) -maytansine (DM3) and N2' -Deacetyl-N2' - (4-mercapto-4-methyl-1-oxopentyl) -maytansine (DM 4).
In a preferred embodiment of the present invention, there is provided a drug conjugate of the antibody as described above, wherein L is1Selected from the group consisting of cleavable linkers, non-cleavable linkers, hydrophilic linkers, pre-charged linkers, and dicarboxylic acid-based linkers.
Preferably, the linker is selected from the group consisting of N-succinimidyl 4- (2-pyridyldithio) valerate (SPP), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), 6-Maleimidocaproyl (MC), Maleimidopropanoyl (MP), valine-citrulline (VC), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and MC-VC-PAB.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the antibody as described above, wherein L is2Is a compound represented by the following formula II:
wherein, X1Selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;
X2selected from alkyl, cycloalkyl and heterocyclyl;
m is 0 to 5; s is a sulfur atom.
Preferably, when X1Is a hydrogen atom, X2When m is 1, the compound shown in the formula II is thioacetic acid S- (3-carbonyl propyl) ester.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the antibody as described above, wherein the small molecule drug is DM1, and the linker L is1Is SMCC, n is 0, thereby forming a drug conjugate of the antibody of formula III below:
in a preferred embodiment of the present invention, there is provided a drug conjugate of the antibody as described above, wherein the small molecule drug is MMAF, and the linker L is1Is MC-VC-PAB, L2Is S- (3-carbonylpropyl) thioacetate, and n is 1, thereby forming a drug conjugate of the antibody represented by the following formula IV:
in a preferred embodiment of the invention, there is provided a drug conjugate of an antibody as described above, in some embodiments the number y (or loading, or DAR) of cytotoxic or small molecule drug conjugated to a single antibody molecule linked to a linker is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, but due to the specificity of the linking reaction the DAR of the small molecule drug conjugated to the antibody linked to a linker is actually an average value between 0 and 10, 1 and 8, 2 and 7, 3 and 6 or 4 and 5. That is, the antibody conjugates of the present invention are actually mixtures of antibodies with different numbers of linker-drugs or just linkers attached, so the y value is the average of the number of drug conjugates and the values are integers or non-integers. In certain embodiments, the drug loading of the ADCs of the present invention ranges from 1 to about 8; from about 2 to about 7; about 3 to about 6; from about 4 to about 5; about 4.1 to about 4.9; about 4.2 to about 4.8; about 4.3 to about 4.7; about 4.4 to about 4.6; about 4.4, 4.6, or about 4.8.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises the VL CDR1 amino acid sequence shown in SEQ ID No. 11 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises a VL CDR1 amino acid sequence as set forth in SEQ ID No. 12 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises a VL CDR2 amino acid sequence as set forth in SEQ ID No. 13 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises the VL CDR1 amino acid sequence shown in SEQ ID No. 22 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises the VL CDR1 amino acid sequence shown in SEQ ID No. 71 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises the VL CDR1 amino acid sequence shown in SEQ ID No. 74 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises the VL CDR1 amino acid sequence shown in SEQ ID No. 23 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises a VL CDR2 amino acid sequence as set forth in SEQ ID No. 24 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region of the anti-CLDN 18.2 antibody comprises a VL CDR3 amino acid sequence as set forth in SEQ ID No. 25 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR1 amino acid sequence shown in SEQ ID No. 15 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR2 amino acid sequence shown in SEQ ID No. 16 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR3 amino acid sequence shown in SEQ ID No. 17 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR1 amino acid sequence shown in SEQ ID No. 18 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR2 amino acid sequence shown in SEQ ID No. 19 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR1 amino acid sequence shown in SEQ ID No. 20 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR2 amino acid sequence shown in SEQ ID No. 21 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR1 amino acid sequence shown in SEQ ID No. 26 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR2 amino acid sequence shown in SEQ ID No. 27 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region of the anti-CLDN 18.2 antibody comprises the VH CDR3 amino acid sequence shown in SEQ ID No. 28 or a mutated sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein said anti-CLDN 18.2 antibody comprises a light chain variable region (VL) comprising the VLCDR1 amino acid sequence shown in SEQ ID NO. 11 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID NO. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID NO. 14 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 12 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 22 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 24 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 25 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 23 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 24 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 25 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 71 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 15 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 74 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 15 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of VHCDR1 shown in SEQ ID No. 15 or a mutant sequence thereof, the amino acid sequence of VH CDR2 shown in SEQ ID No. 16 or a mutant sequence thereof, and the amino acid sequence of VH CDR3 shown in SEQ ID No. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID No. 18 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID No. 16 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID No. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID No. 15 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID No. 19 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID No. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID No. 20 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID No. 21 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID No. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID No. 26 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID No. 27 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID No. 28 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 11 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO. 15 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO. 16 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 12 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO. 15 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO. 16 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 11 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO. 18 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO. 16 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 12 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO. 18 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO. 16 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 11 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO. 15 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO. 19 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 12 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO. 15 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO. 19 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 11 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO:20 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO:21 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO:17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 12 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO:20 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO:21 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO:17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 22 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 24 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 25 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO:26 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO:27 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO:28 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 23 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 24 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 25 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO:26 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO:27 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO:28 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 71 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO. 15 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO. 16 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a VL comprising the VL CDR1 amino acid sequence shown in SEQ ID No. 74 or a mutant sequence thereof, the VL CDR2 amino acid sequence shown in SEQ ID No. 13 or a mutant sequence thereof, and the VL CDR3 amino acid sequence shown in SEQ ID No. 14 or a mutant sequence thereof; and a VH comprising the VH CDR1 amino acid sequence shown in SEQ ID NO. 15 or a mutant sequence thereof, the VH CDR2 amino acid sequence shown in SEQ ID NO. 16 or a mutant sequence thereof, and the VH CDR3 amino acid sequence shown in SEQ ID NO. 17 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody is a murine antibody.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody is a murine antibody, and the murine CLDN18.2 antibody is affinity (affinity) matured to have an affinity increased by 3-10 fold or more, preferably 10 fold or more.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region base sequence of the murine antibody is the base sequence shown in SEQ ID No. 5 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region base sequence of the murine antibody is the base sequence shown in SEQ ID No. 6 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the base sequence of the light chain variable region of the murine antibody is the base sequence shown in SEQ ID No. 5 or a mutant sequence thereof, and the base sequence of the heavy chain variable region is the base sequence shown in SEQ ID No. 6 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region amino acid sequence of the murine antibody is the amino acid sequence shown in SEQ ID No. 7 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region amino acid sequence of the murine antibody is the amino acid sequence shown in SEQ ID No. 8 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region amino acid sequence of the murine antibody is the amino acid sequence shown in SEQ ID No. 7 or a mutant sequence thereof, and the heavy chain variable region amino acid sequence is the amino acid sequence shown in SEQ ID No. 8 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a variable region of a murine antibody and a murine or human antibody constant region comprising a heavy chain constant region of murine IgG1, IgG2a, IgG2b3 or IgG3 and a light chain constant region of kappa or lambda type; the human antibody constant region includes a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, a kappa-or lambda-type light chain constant region, and the like. Preferably, the anti-CLDN 18.2 antibody is a chimeric antibody synthesized from the variable region of a murine antibody and the constant region of a human antibody.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain amino acid sequence of the chimeric antibody is the amino acid sequence represented by SEQ ID No. 9 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain amino acid sequence of the chimeric antibody is the amino acid sequence represented by SEQ ID No. 10 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain amino acid sequence of the chimeric antibody is the amino acid sequence shown in SEQ ID No. 9 or a mutant sequence thereof, and the heavy chain amino acid sequence is the amino acid sequence shown in SEQ ID No. 10 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody is a chimeric antibody, which is matured with affinity (affinity) and the affinity is increased by 3-10 fold or more, preferably 10-fold or more.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody is a humanized antibody.
In a preferred embodiment of the invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region Framework (FR) sequence of the humanized antibody is selected from human germline light chain sequences, e.g., IGKV4-1 x 01(F), IGKV2-28 x 01(F), IGKV2D-28 x 01(F), IGKV1-27 x 01(F), IGKV1-39 x 01(F), IGKV1D-39 x 01(F), IGKV2-40 x 01(F), IGKV2D-29 x 01(F), IGKV2D-40 x 01(F), or IGKV3-15 x 01(F), etc., preferably IGKV4-1 x 01 (F); more preferably, the CDR2 sequence of the selected human germline light chain IGKV4-1 x 01 is identical to SEQ ID NO: 13; examples of the J gene include hJK1, hJK2.1, hJK2.2, hJK2.3, hJK2.4, etc., preferably hJK2.1. The FR sequence preferably comprises a back mutation of 0 to 10 amino acids.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region CDR sequences of the humanized antibody comprising the light chain CDR sequences listed in tables 4-8 or mutated sequences thereof may be defined according to the numbering rules of CCG, Kabat, Chothia, AbM or Contact, respectively.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region sequence of the humanized antibody comprises the amino acid sequence shown in any one of SEQ ID NOs 29 to 33 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the CDR sequences of the light chain variable region of the antibody are optimized for humanization. Preferably, the light chain CDR 10-5 site optimization; preferably, the light chain CDR1 is optimized (mutated) at positions L30E and L34 for the amino acids at the corresponding positions in the human germline CDR 1.
In a preferred embodiment of the invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region Framework (FR) sequence of the humanized antibody is selected from human germline heavy chain sequences, e.g. IGHV1-69 x 02(F), IGHV1-69 x 06(F), IGHV1-69 x 08(F), IGHV1-69 x 09(F), IGHV1-69 x10 (F), IGHV1-69 x 04(F), IGHV1-69 x 14(F), IGHV1/OR15-2 x 02(P), IGHV1-69 x 01(F), IGHV1-69 x 11(F), etc., preferably IGHV1-69 x 01 (F); j genes such as hJh4.1, hJh4.2, hJh4.3, hJh1, hJh2, hJh3.1, hJh3.2, etc., preferably hJh4.1; the FR sequence preferably comprises a back mutation of 0 to 10 amino acids.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region CDR sequences of the humanized antibody comprising the heavy chain CDR sequences listed in tables 4-8 or mutated sequences thereof can be defined according to the numbering rules of CCG, Kabat, Chothia, AbM or Contact, respectively.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region sequence of the humanized antibody comprises the amino acid sequence shown in any one of SEQ ID NOs 34 to 37 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the CDR regions of the antibody are deamination sensitive site-optimized CDR sequences or fragments; preferably, the CDR region deamination sensitive site optimized CDR sequence or fragment is a light chain CDR sequence; preferably, the CDR regions deamination sensitive site optimized CDR sequences or fragments are light chain CDR1 position L30A and/or L30B optimized CDR1 sequences or fragments.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the NS of L30A and/or L30B of the light chain CDR1 of the antibody is mutated to NT, with the proviso that position L30E is not Q and position L34 is not T.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein NS of L30A and/or L30B of the light chain CDR1 of the antibody is mutated to NT, with the proviso that the light chain CDR1 before mutation is the sequence shown in SEQ ID No. 12.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain variable region sequence of the human CDR region deamination sensitive site-optimized CDR sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs 46 to 49 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the CDR regions of the antibody are deamination sensitive site-optimized CDR sequences or fragments; preferably, the CDR region deamination sensitive site optimized CDR sequence or fragment is a heavy chain CDR sequence; preferably, the CDR regions deamination sensitive site optimized CDR sequences or fragments are H99 and/or H100 optimized CDR sequences or fragments of heavy chain CDR 3.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the NS at position H99 and/or H100 of the heavy chain CDR3 of the antibody is mutated to NT, with the proviso that L30E of the light chain CDR1 is not Q and L34 is not T.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the NS at position H99 and/or H100 of the heavy chain CDR3 of the antibody is mutated to NT, with the proviso that the light chain CDR1 before the mutation is the sequence shown in SEQ ID No. 12.
Wherein the light chain CDR1 positions L30A, L30B, L30E and L34 and the heavy chain CDR3 positions H99 and H100 are defined by Kabat numbering convention as described above.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain variable region sequence of the human CDR region deamination-sensitive site-optimized CDR sequence comprises an amino acid sequence as set forth in any one of SEQ ID NOs 50 to 54 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a combination of a light chain variable region having an amino acid sequence represented by any one of SEQ ID NOs 29 to 33 or SEQ ID NOs 46 to 49 or a mutant sequence thereof and a heavy chain variable region having an amino acid sequence represented by any one of SEQ ID NOs 34 to 37 or SEQ ID NOs 50 to 54 or a mutant sequence thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the light chain of the humanized antibody comprises a light chain constant region selected from human antibodies of the kappa or lambda type or variants thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain of the humanized antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4, or a variant thereof.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain of the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4, or a variant thereof, and the heavy chain constant region or variant thereof comprises a mutation at position 243, or at positions 239, 330 and 332 of the human IgG1Fc region.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the heavy chain of the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 or a variant thereof, and the heavy chain constant region or variant thereof comprises a variant of the human IgG1Fc region at positions 356-358 of EEM or DEL.
In a preferred embodiment of the present invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the light chain of the humanized and/or CDR region deamination sensitive site optimized antibody comprises the amino acid sequence shown in SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 45, SEQ ID NO 56, SEQ ID NO 58 or SEQ ID NO 60 or a light chain sequence having at least 85% sequence homology thereto. The light chain sequence is a full-length light chain sequence or a light chain variable region sequence. Said at least 85% sequence homology is preferably at least 90% sequence homology; more preferably at least 95% sequence homology; most preferably at least 99% sequence homology.
In a preferred embodiment of the invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the heavy chain of the antibody, which is humanized and/or CDR regions are site-optimized for deamination sensitivity and/or preferably IgG1 variant, comprises the amino acid sequence shown in SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 62 or SEQ ID NO 63 or a heavy chain sequence having at least 85% sequence homology thereto. The heavy chain sequence is a full-length heavy chain sequence or a heavy chain variable region sequence. Said at least 85% sequence homology is preferably at least 90% sequence homology; more preferably at least 95% sequence homology; most preferably at least 99% sequence homology.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 38 and the amino acid sequence shown as SEQ ID NO. 39.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 40 and the amino acid sequence shown in SEQ ID NO. 39.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 38 and the amino acid sequence shown as SEQ ID NO. 41.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 40 and the amino acid sequence shown in SEQ ID NO. 41.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 42 and the amino acid sequence shown as SEQ ID NO. 39.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 42 and the amino acid sequence shown in SEQ ID NO. 43.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 42 and the amino acid sequence shown as SEQ ID NO. 44.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 45 and the amino acid sequence shown in SEQ ID NO. 43.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 45 and the amino acid sequence shown as SEQ ID NO. 44.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the humanized antibody comprises a humanized light and heavy chain combination, preferably the humanized light and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 45 and the amino acid sequence shown as SEQ ID NO. 39.
In a preferred embodiment of the present invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a humanized, and/or site-optimized light chain and heavy chain combination of CDR regions that are deaminated sensitive, preferably the light chain and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 38 and the amino acid sequence shown in SEQ ID NO. 55.
In a preferred embodiment of the present invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a humanized, and/or site-optimized light chain and heavy chain combination of CDR regions that are deaminated sensitive, preferably the light chain and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 42 and the amino acid sequence shown in SEQ ID NO. 55.
In a preferred embodiment of the present invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a humanized, and/or site-optimized light chain and heavy chain combination of CDR regions that are deaminated sensitive, preferably the light chain and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 56 and the amino acid sequence shown in SEQ ID NO. 57.
In a preferred embodiment of the present invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a humanized, and/or site-optimized light chain and heavy chain combination of CDR regions that are deaminated sensitive, preferably the light chain and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 58 and the amino acid sequence shown in SEQ ID NO. 57.
In a preferred embodiment of the invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a light chain and heavy chain combination of humanization, and/or CDR region deamination-sensitive site optimization, and/or IgG Fc variants, preferably the light chain and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 38 and the amino acid sequence shown as SEQ ID NO. 59.
In a preferred embodiment of the invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a light chain and heavy chain combination of humanization, and/or CDR region deamination-sensitive site optimization, and/or IgG Fc variants, preferably the light chain and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 42 and the amino acid sequence shown as SEQ ID NO. 59.
In a preferred embodiment of the invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a light chain and heavy chain combination of humanization, and/or CDR region deamination-sensitive site optimization, and/or IgG Fc variants, preferably the light chain and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 60 and the amino acid sequence shown in SEQ ID NO. 61.
In a preferred embodiment of the invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a light chain and heavy chain combination of humanization, and/or CDR region deamination-sensitive site optimization, and/or IgG Fc variants, preferably the light chain and heavy chain combination is: the amino acid sequence shown as SEQ ID NO. 60 and the amino acid sequence shown as SEQ ID NO. 62.
In a preferred embodiment of the invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the antibody comprises a light chain and heavy chain combination of humanization, and/or CDR region deamination-sensitive site optimization, and/or IgG Fc variants, preferably the light chain and heavy chain combination is: the amino acid sequence shown in SEQ ID NO. 60 and the amino acid sequence shown in SEQ ID NO. 63.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody is a humanized antibody, a CDR sequence-optimized antibody, a CDR region deamination sensitive site-optimized, an IgG Fc variant; the antibodies are affinity matured with 3-10 fold or more, preferably 10 fold or more, increase in affinity.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody comprises a half-antibody or an antigen-binding fragment of a half-antibody, preferably, a Fab, Fab ', F (ab')2, Fv or single chain Fv fragment (scFv).
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, which is an antibody drug conjugate represented by the following formula V:
or, an antibody drug conjugate of formula VI below:
or, an antibody drug conjugate of formula VII below:
or, an antibody drug conjugate of formula VIII below:
wherein Ab10 comprises a light chain as shown in SEQ ID NO. 38 and a heavy chain as shown in SEQ ID NO. 39; the Ab6 contains a light chain as shown in SEQ ID NO. 42 and a heavy chain as shown in SEQ ID NO. 39.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the antibody has a strong drug conjugate (ADC) and human CLDN 18.2-specific binding capacity, and has an affinity of nM, preferably less than 100pM, more preferably less than 20pM, and most preferably 10pM, as measured by the KinExA method.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of the anti-CLDN 18.2 antibody induces ADCC activity between human blood cells (effector cells) and tumor cells, enhancing killing of the tumor cells by ADCC.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of the anti-CLDN 18.2 antibody induces CDC activity between human blood cells (effector cells) and tumor cells, enhancing killing of the tumor cells by CDC.
In a preferred embodiment of the present invention, there is provided a drug conjugate of an anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of an anti-CLDN 18.2 antibody induces apoptosis of CLDN 18.2-positive tumor cells, showing that the CLDN18.2 antibody of the present invention induces apoptosis of CLDN 18.2-positive tumor cells.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of the anti-CLDN 18.2 antibody inhibits growth of CLDN 18.2-positive tumor cells, showing an inhibitory effect of the CLDN18.2 antibody of the present invention on growth of CLDN 18.2-positive tumor cells.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of the anti-CLDN 18.2 antibody inhibits growth of CLDN 18.2-positive tumor cells in vivo, showing in vivo efficacy of the CLDN18.2 antibody of the present invention.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of the anti-CLDN 18.2 antibody inhibits growth of CLDN 18.2-overexpressing tumor cells in vivo, showing in vivo efficacy of the CLDN18.2 antibody of the present invention.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of the anti-CLDN 18.2 antibody binds to murine CLDN 18.2.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of the anti-CLDN 18.2 antibody and murine CLDN18.1 do not bind.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the drug conjugate of the anti-CLDN 18.2 antibody exhibits excellent in vivo drug metabolism (PK) performance, particularly, good in vivo half-life (T1/2).
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, wherein the anti-CLDN 18.2 antibody has bispecific antibody molecule activity.
In a preferred embodiment of the present invention, there is provided a drug conjugate of the anti-CLDN 18.2 antibody as described above, comprising a Chimeric Antigen Receptor (CAR) T, NK cell activity of the anti-CLDN 18.2 antibody.
The present invention further provides a method for producing a drug conjugate of an antibody, wherein,
when n is 1, the preparation method comprises the following steps:
(1)preparation of intermediate 1: taking the antibody and the linker L2Mixing the two solutions, reacting and purifying to obtain a solution containing an intermediate 1, wherein the intermediate 1 is shown as a formula IX below:
wherein, X1Selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;
X2selected from alkyl, cycloalkyl and heterocyclyl;
m is 0 to 5; s is a sulfur atom;
preferably, X1Is a hydrogen atom, X2Is alkyl, m is 1, i.e. L2Is thioacetic acid S- (3-carbonylpropyl) ester;
(2) preparation of intermediate 2: taking out the joint L1And the drug is prepared into an intermediate 2: l is1-D;
(3) Mixing the solution containing the intermediate 1 obtained in the step (1) with the solution containing the intermediate 2 obtained in the step (2), reacting and purifying to obtain a solution of a drug conjugate containing the antibody;
when n is 0, the preparation method comprises the following steps:
(1) preparation of intermediate 3: taking the antibody and the linker L1Mixing the mixture in the solution, reacting and purifying to obtain a solution containing an intermediate 3;
(2) and (2) mixing the solution containing the intermediate 3 obtained in the step (1) with the solution containing the drug, reacting, and purifying to obtain the solution of the drug conjugate containing the antibody.
In a preferred embodiment, in the above steps (1) and/or (2), the reaction temperature is 25 ℃; the reaction time is 2-4 hours.
In a preferred embodiment, the purification is purification by gel filtration.
In a preferred embodiment, in the above steps (1) and/or (2), the reaction temperature is 25 ℃; the reaction time is 2-4 hours; and the purification is purification by gel filtration. More preferably Sephadex G25 gel column desalting purification.
The present invention further provides a pharmaceutical composition, which, in a preferred embodiment of the present invention, comprises a drug conjugate of the above CLDN18.2 antibody, and optionally a pharmaceutically acceptable carrier, excipient and/or stabilizer. Preferably, the pharmaceutical composition further comprises an immune checkpoint antibody.
In a preferred embodiment of the present invention, the present invention further provides a method for treating cancer, comprising administering the above CLDN18.2 antibody or pharmaceutical composition to a subject in need thereof in an amount effective to treat cancer; preferably, the method comprises administering any combination comprising a CLDN18.2 antibody of the invention, more preferably, a combination comprising a CLDN18.2 antibody and an immune checkpoint antibody, comprising a PD-1 antibody or an antibody drug as described above.
The present invention further provides a method of treating cancer comprising a method of combination therapy with the anti-CLDN 18.2 antibody of the present invention.
In a preferred embodiment, the present invention further provides a method of treating cancer, preferably lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, leukemia, etc., or metastatic lesions of cancer, by administering the drug conjugate of CLDN18.2 antibody or the pharmaceutical composition containing the same as described above.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention. The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the invention initiates an Antibody Drug Conjugate (ADC) directed against CLDN 18.2. Wherein the antibody molecule is a humanized antibody, the specific binding activity with human and mouse CLDN18.2 is better, and the Emax is higher; high affinity (KinExA) (reaching the 10pM level); CDC activity is better in human blood cells; better apoptosis activity of the CLDN18.2+ cell, better activity of inhibiting the growth of tumor cells, better drug effect of animals, and drug metabolism (PK) in vivo, in particular longer T1/2. And murine CLDN18.2 provides a better choice of non-primate experimental animals for preclinical studies. The prominent PK can produce the same or better drug effect at low dose, and has the advantage of low production cost. ADC molecules obtained by coupling the antibody and cytotoxin not only retain the characteristics of excellent specific binding activity and endocytosis activity of the antibody and mouse CLDN18.2 and high-efficiency killing of tumor cells, but also carry cytotoxic toxin, can specifically and targetedly kill the tumor cells, specifically inhibit the proliferation of the tumor cells in a targeted manner, and generate unexpected more excellent tumor treatment efficacy. These features allow the ADC drugs of the present invention, as well as pharmaceutically acceptable salts, solvates or combinations thereof in combination with other drugs, to provide more specific and effective, better treatment options, means and methods for tumor patients, particularly CLDN18.2 positive cancer patients. In addition, the antibody drug conjugate/antibody provided by the invention has better pharmacokinetic performance, large safety window and lower toxic and side effects.
Drawings
Fig. 1 is a binding activity (ELISA) of murine anti-human CLDN18.2 antibody mab5b and human CLDN18.2 (fig. 1a) and murine CLDN18.2 (fig. 1b) of the present invention;
FIG. 2is a graph of the binding activity (ELISA) of the humanized antibody of anti-human CLDN18.2 antibody mab5b (FIG. 2a) and the humanized optimized antibody (FIG. 2b) and human CLDN18.2 of the invention;
FIG. 3 shows the sequence optimization of deamidation sensitive (deamidation) site of the antibody of the present invention. Figure 3a, CDR1 sequence KSSQSLLNSGNQKNYLT, heavy chain H100S- > T mutation with loss of binding activity (Ab 30); FIG. 3b, CDR1 sequence KSSQSLLNSGNQKNYLT, light chain L30B S- > T and heavy chain H100S- > T mutations inactivated (Ab 35);
FIG. 4 is an evaluation of CDC activity of a humanized anti-human CLDN18.2 antibody, humanized preferred antibody of the present invention;
FIG. 5 is the activity of the humanized anti-human CLDN18.2 antibody, humanized preferred antibody, of the present invention to induce apoptosis of tumor cells (FIG. 5a, FIG. 5 b);
FIG. 6 is an in vivo efficacy evaluation of a humanized anti-human CLDN18.2 antibody, a humanized preferred antibody animal model of the present invention (FIG. 6a, FIG. 6 b);
FIG. 7 shows the analysis of the endocytic activity of the humanized antibodies Ab10 and Ab6 of the present invention;
FIG. 8 shows the detection of the endocytosis activity of Ab10 antibody cell drug conjugate ADC1 and ADC 2;
fig. 9 shows that Ab10 antibody cell drug conjugate ADC1 (fig. 9a) and ADC2 (fig. 9b) inhibit the proliferation activity of target cells.
Detailed Description
Interpretation of terms:
in order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The three letter and one letter codes for amino acids used in the present invention are known to those skilled in the art or described in j.biol.chem,243, p3558 (1968).
The term "CLDN 18.2" includes isoforms, mammalian (e.g., human) CLDN18.2, species homologs of human CLDN18.2, and analogs comprising at least one common epitope with CLDN 18.2. The amino acid sequence of CLDN18.2 (e.g., human CLDN18.2) is known in the art as shown in the NCBI database.
The term "CLDN 18.1" includes isoforms, mammalian (e.g., human) CLDN18.1, species homologs of human CLDN18.1, and analogs comprising at least one common epitope with CLDN 18.1. The amino acid sequence of CLDN18.1 (e.g., human CLDN18.1) is known in the art as shown in the NCBI database.
The "antibody" or "antibody molecule" of the invention may be an antibody or binding fragment thereof. The "binding fragment" may be an antigen or an antigenic epitope binding fragment. The "CLDN 18.2 antibody", "anti-CLDN 18.2 antibody", "CLDN 18.2 antibody molecule" and "anti-CLDN 18.2 antibody molecule" described herein are used interchangeably.
As used herein, the terms "drug conjugate," "conjugate," or "ADC" of an antibody are used interchangeably to refer to a drug conjugate of an antibody having the structure shown in formulas I, III, IV, V, VI, or VII, etc.
The term "drug", "small molecule drug", "cytotoxic agent", "toxin" or "drug moiety" refers to a substance that inhibits or prevents the expression activity of a cell, the function of a cell and/or causes destruction of a cell, the term is intended to include drugs, cytotoxic agents, pro-apoptotic agents, toxins, tubulin inhibitors, nucleases (including dnases and rnases), hormones, immunomodulators, integrins, pergolds, photosensitizers or dyes, radioisotopes or radionuclides, oligonucleic acids, interfering RNAs, peptides, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins or peptides, or combinations thereof examples of cytotoxic agents include, but are not limited to, auristatin, chlortetracycline (maytansine) class DM1, DM3 or DM4, ricin (ricin), ricin a chain, combretastatin (combretastatin), duocarmycin (rabbit toxin), dolicheotoxin (doxorubicin), phytotoxin (e.g. phytotoxin), phytotoxin (e.g. a), phytotoxin (e.g. luteolin), phytotoxin (e.g. phytotoxin), phytotoxin (e.g. luteolin), phytotoxin (e.g. lectin), phytotoxin (e.g. luteolin), phytotoxin (e.g. luteolin (e.g. lectin), phytotoxin (e.g. lectin), phytotoxin (e.g. toxin (e.g. lectin), phytotoxin (e.g. phytotoxin), phytotoxin (e.g. toxin), phytotoxin (e.g. lectin), phytotoxin (e.g.
"tubulysins" refers to a class of compounds that interfere with the mitotic process of cells by inhibiting or promoting the polymerization of tubulin, thereby exerting an anti-tumor effect. Non-limiting examples thereof include: maytansinoids, calicheamicin, taxus anhui, vincristine, colchicine, urodoline/auristatin.
Auristatins are fully synthetic drugs whose chemical structure is relatively easy to modify in order to optimize their physical properties and drug-forming properties. The auristatin derivatives for antibody coupling mainly include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), the former is synthesized by adding 2-amino-1-phenylpropyl-1-ol to C-terminal of synthetic pentapeptide derived from natural tubulin polymerase inhibitor Dolatatin 10 (dolastatin-10). The inhibitory activity of MMAE on various human tumor cell strains is less than one nanomole. In order to reduce the cytotoxic activity of MMAE, a phenylalanine is added at the C-terminal of the Dolabellin 10 by MMAF, because a carboxyl is introduced into the structure, the cell membrane permeability of MMAF is poor, so that the biological activity of MMAF on cells is obviously reduced, but the inhibitory activity of MMAF on cells after being coupled with an antibody is greatly improved (US 7750116).
In some embodiments, the antibody cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof comprises an antibody of the invention coupled to one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that do not function by inhibiting tubulin polymerization. Maytansine was originally isolated from the east African shrub Maytenus serrata (Maytenus serrata) (U.S. Pat. No.3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol vinegar (U.S. Pat. No.4,151,042). Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they: (i) relatively easy to prepare by fermentation or chemical modification or derivatization of the fermentation product; (ii) are readily derivatized with functional groups suitable for coupling to antibodies via non-disulfide linkers; (iii) is stable in plasma; and (iv) is effective against a variety of tumor cell lines. Maytansinoid compounds suitable for use as maytansinoid drug moieties are well known in the art and may be isolated from natural sources according to known methods or produced using genetic engineering techniques (see Yu et al (2002) PNAS 99: 7968-. Maytansinol and maytansinol analogues can also be prepared synthetically according to known methods. Exemplary embodiments of maytansinoid drug moieties include: DM1, DM3, and DM4, as disclosed herein.
The terms "linker unit", "linker" and "linker" as used herein refer to groups suitable for use in the present invention for linking the antibodies and small molecule drugs of the present invention. Exemplary linkers include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4- (2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). In one embodiment, the linker is MC-vc-PAB.
In the following description, either a cleavable linker or a non-cleavable linker is included. The cleavable linker may be a chemically and enzymatically labile linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker. Enzyme-labile linkers are widely chosen as cleavable linker candidates in ADCs due to high plasma stability and good intracellular cleavage selectivity and efficiency. In some embodiments, the enzyme labile linker may comprise peptide units (AAs), or amino acid units, including but not limited to dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides are selected from the group consisting of valine-citrulline (Val-Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg), phenylalanine-citrulline (Phe-Cit), phenylalanine-lysine (Phe-Lys), and phenylalanine-arginine (Phe-Arg). Typical enzyme labile linkers include Val-Cit and Phe-Lys, which are recognized by cathepsin B. Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine (g1y-g1y-g1 y). The amino acid units can comprise naturally occurring amino acid residues, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid units can be designed and optimized in their selectivity for enzymatic cleavage by specific enzymes such as tumor-associated proteases, cathepsin B, C and D, or plasma proteases.
Drug Loading (Loading) is represented by y, the average number of drug modules per antibody in the molecules of formulae I, III, IV, V and VI, also known as the drug antibody conjugation ratio (DAR). The average number of drug modules per antibody in the ADC preparation from the conjugation reaction can be characterized by conventional means, such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC in y can also be determined. In some cases, separation, purification, and characterization of homogeneous ADCs where y is a certain value from ADCs with other drug loadings may be accomplished by means such as reverse phase HPLC or electrophoresis. The drug load may range from 0.8 to 10 drug moieties (D) per antibody. In certain embodiments, higher drug loadings, e.g., y >5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody drug conjugates. In certain embodiments of the invention, the drug loading ranges from 1 to about 9; from about 2 to about 8; about 3 to about 6; from about 4 to about 5; about 4.1 to about 4.9; about 4.2 to about 4.8; about 4.3 to about 4.7; about 4.4 to about 4.6; or about 4.4 or 4.8.
In certain embodiments, less than the theoretical maximum of drug moieties are conjugated to the antibody in a conjugation reaction. The antibody may comprise, for example, lysine residues that are not reactive with the drug linker intermediate or linker reagent, as discussed below. Only the most reactive lysine groups can react with the limb-reactive linker reagent. In general, antibodies do not contain many free and reactive cysteine thiol groups, to which a drug moiety can be attached: in fact, most cysteine thiol groups in antibodies exist as disulfide bridges. In certain embodiments, the antibody may be reduced with a reducing agent such as Dithiothreitol (DTT) or Tricarbonylethylphosphine (TCEP) under partially or fully reducing conditions to produce reactive cysteine thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups, such as lysine or cysteine.
The loading of the ADC (drug/antibody ratio DAR) can be controlled in different ways, for example by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) cysteine thiol-modified moieties or limiting reducing conditions, (iv) engineering the amino acid sequence of the antibody by recombinant techniques such that the number and position of cysteine residues is altered in order to control the number and/or position of linker-drug attachments (such as thioMab or thioFab prepared as described herein and in W02006/034488 (incorporated herein by reference in its entirety)).
It will be appreciated that if more than one nucleophilic group is reacted with a drug linker intermediate or with a linker reagent and a subsequent drug moiety reagent, the resulting product is a mixture of ADC compounds having a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay specific for the antibody and specific for the drug. The various ADC molecules in the mixture can be identified by mass spectrometry and separated by HPLC, for example hydrophobic interaction chromatography. In certain embodiments, homogeneous ADCs having a single loading value may be separated from the coupling mixture by electrophoresis or chromatography.
The methods, compositions, and combination therapies described herein may be combined with other active agents or therapeutic modalities, the method comprises administering to a subject an anti-CLDN 18.2 antibody molecule of the invention, optionally, with PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, Tim-3 antibodies (immunotherapy) or other tumor therapy antibodies, Her-2, EGFR, VEGF, VEGFR antibodies, and the like, and one or more inhibitors of ADC (antibody drug conjugate, such as T-DM1), bispecific antibodies, chemotherapeutic drugs, and the like, further comprising administering an anti-CLDN 18.2 antibody molecule, an additional active agent, or all may be administered in such amounts or doses, the amount or dose is higher, lower or equal to the amount or dose of each active agent used alone (e.g., as a monotherapy). The amount or dose of the anti-CLDN 18.2 antibody, additional active agent or all administered is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose of each active agent used alone (e.g., as monotherapy).
The terms "polypeptide", "peptide" and "protein" (if single-chain) are used interchangeably herein. The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. The term "isolated" refers to a substance that is removed from its original or original environment (e.g., the natural environment if it occurs in nature). A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, serine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), self-branching side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).
The term "antibody" as used herein includes immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds, the constant regions of the heavy chains of immunoglobulins have different amino acid compositions and sequences and thus are different in antigenicity, and accordingly, the immunoglobulins can be classified into five classes, or isotypes, called immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains thereof are μ chain, δ chain, γ chain, α chain, and ε chain, the same class of igs can be classified into different subclasses depending on the differences in the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chains, e.g., iggs can be classified into IgG1, IgG2, IgG3, and IgG 4.
In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa or lambda chain or a variant thereof. In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, 2, 3, 4 or variants thereof.
The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3.
The CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the invention are in numbers and positions conforming to known Kabat, Contact, CCG, AbM, and Chothia numbering rules. The boundaries of a given CDR may vary depending on the scheme used for identification. The rules of definition and the CDR sequences defined by the antibody are shown in tables 3-8. For example, the Kabat approach is based on structural alignment, while the Chothia approach is based on structural information. The numbering used for the Kabat and Chothia schemes is based on the most commonly used antibody region sequence lengths, and the inserts (e.g., "30 a") and some antibodies are adapted by insertion of letters to present deletions. Both protocols placed certain inserts and deletions ("indels") at different positions resulted in differential numbering. The Contact protocol is based on analysis of the complex crystalline structure and is similar in many respects to the Chothia numbering protocol. Thus, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) and the individual CDRs of that antibody or region thereof (e.g., "CDR-H1, CDR-H2) are to be understood as encompassing complementarity determining regions as defined by any of the above-described known schemes described herein. In some cases, schemes are specified for identifying one or more particular CDRs, e.g., CDRs as defined by the Kabat, Chothia, or Contact methods. In other cases, a particular amino acid sequence of a CDR is given.
The term "murine antibody" is in the present invention a monoclonal antibody to human CLDN18.2 prepared according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with the CLDN18.2 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine CLDN18.2 antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or IgG4 or variant thereof.
The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized antibodies").
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. Establishing a chimeric antibody, selecting and establishing a hybridoma secreting a murine monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of the obtained human antibody according to needs, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a vector, and finally expressing the chimeric antibody molecule in a eukaryotic cell, an industrial system or a prokaryotic industrial system. In a preferred embodiment of the present invention, the antibody light chain variable region of said CLDN18.2 chimeric antibody further comprises a light chain FR region of murine kappa, lambda types or variants thereof. The antibody heavy chain variable region of the CLDN18.2 chimeric antibody further comprises a heavy chain RF region of murine 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 IgG1 or IgG4 heavy chain constant region, or IgG1 using amino acid mutations that alter ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement dependent cytotoxicity) activity. ADCC and CDC effector functions of the antibody can be reduced or eliminated or enhanced by modifying the Fc segment of IgG. The modification refers to mutation in the heavy chain constant region of an antibody, such as N297A, L234A, L235A selected from IgG 1; IgG2/4chimera, F235E, L234A/E235A, F243L, or S239D/A330L/I332E mutations of IgG 4.
The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting a mouse CDR sequence into a human antibody variable region framework. Specifically, the CDR of the CLDN18.2 antibody of the invention is an antibody produced by grafting each CDR sequence defined by the numbering rules of CCG, Kabat, AbM, Chothia or Contact into the framework of the human antibody variable region. Preferably, 0 to 5 positions of the CDR, preferably the light chain CDR1, of the CLDN18.2 antibody of the invention are mutated to the amino acids at the corresponding positions of the CDRs of an adult antibody. These can overcome the strong antibody variable antibody response induced by chimeric antibodies due to the large number of mouse protein components. Human FR germline sequences are available from websites www.imgt.org and www.vbase2.org of ImmunoGeneTiCs (IMGT).
In a preferred embodiment of the invention, the CDR sequences of the humanized antibody mouse of CLDN18.2 are selected from the group consisting of SEQ ID NOS: 11-28. Human antibody variable region frameworks were selected by design, wherein the light chain FR region sequences on the antibody light chain variable regions, derived from the combined sequences of human germline light chains IGKV4-1 × 01(F) and hJK2.1 SEQ ID NOS: 29-33, comprising the FR1, FR2, FR3 regions of human germline light chains IGKV4-1 × 01(F) and the FR4 region of hJK2.1; wherein the heavy chain FR region sequence in the heavy chain variable region of the antibody is derived from the combined sequence of human germline heavy chains IGHV1-69 x 01(F) and hJH4.1 SEQ ID NO:34-37, and comprises FR1, FR2, FR3 regions of human germline heavy chains IGHV1-69 x 01(F) and FR4 region of hJH 4.1. To avoid reduced immunogenicity and resulting reduced activity, the human antibody variable regions may be subjected to minimal back-mutations to maintain activity. In a preferred embodiment of the invention, the humanized antibody variable region is back mutated to 0, i.e., a fully humanized antibody.
The term "deamination" refers to the removal of an amino group at a site or at a site on a molecule. A "deamination-sensitive site" refers to a molecule and a site on a molecule that is easier and more prone to deamination.
The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed as activity on a molar basis. Preferably, the antibody fragment retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to: fab, Fab ', F (ab')2, Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) nat. Biotechnol.23: 1126-.
A "Fab fragment" consists of one light and one heavy chain of CH1 and the variable domains. 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 CH1 and CH2 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 ')2A molecule. "F (ab')2Fragments"heavy chains comprising two light chains and two portions comprising a constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, F (ab')2The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region.
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, diabodies (diabodies), bispecific diabodies and triabodies (triabodies), antibody fragments that have been covalently or non-covalently linked together, and the like.
Antibody molecules include diabodies (diabodies) and single chain molecules, as well as antigen-binding fragments of antibodies (e.g., Fab, F (ab')2, scFv, and Fv). Antibody molecules comprise or consist of one heavy chain and one light chain (referred to herein as half-antibodies). Fab ', F (ab')2, Fc, Fd, Fv, single chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (diabodies and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modifying whole antibodies, or those antibody molecules synthesized de novo using recombinant DNA techniques. These functional antibody fragments retain the ability to selectively bind to their corresponding antigen or receptor. Antibodies and antibody fragments can be from any antibody class, including but not limited to IgG, IgA, IgM, IgD, and IgE and from any antibody subclass (e.g., IgG1, IgG2, IgG3, and IgG 4). Antibody molecules can be prepared either monoclonal or polyclonal. The antibody may also be a human antibody, a humanized antibody, a CDR-grafted antibody or an in vitro generated antibody. The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG 4. The antibody may also have a light chain, for example selected from the K or λ types. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" in the present invention.
The antibodies disclosed herein may also be single domain antibodies. Single domain antibodies may include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking a light chain, single domain antibodies derived from conventional 4 chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any antibody of the prior art, or any single domain antibody in the future. Single domain antibodies may be derived from any species, including but not limited to mouse, human, camel, alpaca, fish, heavy fish, goat, rabbit, and cow. According to some aspects, the single domain antibody is a naturally occurring single domain antibody, referred to as a heavy chain antibody lacking a light chain. For clarity reasons, such variable domains derived from heavy chain antibodies naturally lacking a light chain are referred to herein as VHHs or nanobodies to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules may be derived from antibodies raised in Camelidae (Camelidae) species (e.g. camel, alpaca, dromedary, camel and guanaco). Other species than camelids may produce heavy chain antibodies that naturally lack a light chain, and such VHHs are also contemplated. The VH and VL regions can be subdivided into hypervariable regions, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions, termed "framework regions" (FRs). The framework regions and the extent of the CDRs have been defined in a number of ways.
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. The host cell of the vector of the present invention may be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, 293T, 293E, BHK cells, suitable insect cells include, but are not limited to, Sf9 cells.
Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques. Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the antibody experimental technical guidelines of cold spring harbor. Antigen-binding fragments can likewise be prepared by conventional methods.
By "treating" is meant administering a therapeutic agent, such as a composition comprising any of the binding compounds of the invention, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient.
In the present invention, the "pharmaceutical composition" comprises an effective amount of an ADC according to the invention (as active ingredient), together with at least one pharmaceutically acceptable carrier, diluent or excipient. In preparation, the active ingredient is typically mixed with, or diluted with, excipients or enclosed within a carrier which may be in the form of a capsule or sachet. When the excipient serves as a diluent, it may employ a solid, semi-solid or liquid material as a vehicle, carrier or medium for the active ingredient. Thus, the composition may be a solution, a sterile injectable solution, or the like.
An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.
"homology", "variant sequences", "mutation" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology. "optimization" refers to a mutation that maintains or improves the binding of the antibody to the antigen, and in the present invention, refers to a mutation that maintains, maintains or improves the binding to CLDN 18.2.
As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.
As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a particular portion of nucleic acid, RNA and/or DNA are amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. The PCR used in the present invention is considered as one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which method comprises using a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific portion of the nucleic acid.
"optional," "optionally," "any," or "any" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1 antibody heavy chain variable region" means that the antibody heavy chain variable region of a particular sequence may, but need not, be present.
As used herein, "a" and "an" are used herein to refer to one or more grammatical objects. The term "or" is used herein to mean and is used interchangeably with the term "and/or" unless the content clearly dictates otherwise. "about" and "approximately" shall generally mean an acceptable degree of error in the measured quantity in view of the nature or accuracy of the measurement. Exemplary degrees of error are typically within 10% thereof and more typically within 5% thereof. The methods and compositions disclosed herein encompass polypeptides and nucleic acids having a specified sequence, variant sequence, or sequence that is substantially identical or similar thereto, e.g., a sequence that is at least 85%, 90%, 95%, 99% or more identical to the specified sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence.
"pharmaceutical composition" means a mixture comprising one or more compounds of the invention or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity. The therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high antibody concentrations. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
Suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc.; the formulation may further comprise: wetting agents, emulsifiers, preservatives (such as methyl and propyl hydroxybenzoates), and the like. The antineoplastic drug may be formulated in unit or multi-unit dosage forms, each dosage form containing a predetermined amount of the ADC of the invention calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients.
Furthermore, as described in the examples of the present invention, the anti-CLDN 18.2 antibody and the drug conjugate of CLDN18.2 antibody can bind to CLDN18.2 to induce apoptosis of target cells (tumor cells), inhibit tumor cell growth, increase ADCC and CDC killing of effector cells on tumor cells in vivo, thereby achieving the purpose of treating cancer patients. Thus, in certain embodiments, the anti-CLDN 18.2 antibody and the drug conjugate of a CLDN18.2 antibody described herein exhibit anti-tumor effects of the antibody of the present invention by these mechanisms, and a method of inhibiting tumor cell growth, comprising administering to a subject a therapeutically effective amount of the anti-CLDN 18.2 antibody and the drug conjugate of a CLDN18.2 antibody described herein. The method is suitable for in vivo treatment of cancer. To obtain a target specific therapeutic effect, the anti-CLDN 18.2 antibody molecule may be administered together with other antibodies. When a CLDN18.2 antibody and a drug conjugate of a CLDN18.2 antibody are administered in combination with one or more active agents, the combination may be administered in any order or simultaneously to a cancer type, in particular a tumor patient with high expression of CLDN 18.2. In certain aspects, methods of treating (e.g., reducing or ameliorating) a hyperproliferative condition or disease (e.g., cancer) in a subject, e.g., a solid tumor, a hematologic cancer, a soft tissue tumor, or a metastatic lesion, are provided. The method comprises administering to the subject one or more anti-CLDN 18.2 antibodies or drug conjugates of CLDN18.2 antibodies of the invention, either alone or in combination with other active agents or therapeutic modalities.
Combinations further include inhibitors or activators of immune checkpoint modulators, e.g., anti-PD-L1 antibody molecules, anti-PD-1 antibody molecules, or CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies), or inhibitors or activators of non-immune checkpoint modulators (e.g., chemical drugs, small molecule targeted drugs, macromolecules including antibody targeted drugs, such as anti-Her2, anti-VEGF, anti-VEGFR, anti-EGFR, etc., antibody conjugated drugs, bispecific antibodies, CAR-T cell combinations, etc.), or any combination thereof. CLDN18.2 antibody therapy can also be combined with standard cancer therapy. Antibody toxin conjugate therapy may be effectively combined with a chemotherapeutic regimen. In these cases, the dose of chemotherapeutic agent administered may be reduced. The composition can be administered in combination with one or more, an immunomodulator (e.g., an activator of a costimulatory molecule or an inhibitor of an inhibitory molecule); vaccines or other forms of cellular immunotherapy.
The term "immune checkpoint" refers to a set of molecules on the cell surface of an immune cell that can act as a "gate" to down-regulate or suppress an immune response, such as an anti-tumor immune response, in turn treating a tumor in combination with an antibody of the invention. Immune checkpoint molecules include, but are not limited to, PD-1, PD-L1, cytotoxic T lymphocyte antigen 4(CTLA-4), B7-H1, B7-H3, OX-40, 4-1BB (CD137), CD40, and lymphocyte activation gene 3(LAG-3), among others.
anti-CLDN 18.2 antibody molecules, alone or in combination with another immunomodulator (e.g., anti-LAG-3, anti-Tim-3, anti-PD-L or anti-PD-L1, anti-CTLA-4 antibody molecules), are used to treat gastric, pancreatic, lung, esophageal, ovarian cancer, and the like. The anti-CLDN 18.2 antibody molecule may be administered in combination with one or more of: immune-based strategies, targeted drugs (e.g., VEGF inhibitors such as monoclonal antibodies against VEGF); VEGF tyrosine kinase inhibitors such as sunitinib, sorafenib, apatinib; RNAi inhibitors or inhibitors of downstream mediators of VEGF signaling, e.g., inhibitors of the mammalian target of rapamycin (mTOR).
As used herein, the terms "cancer", "cancer patient" are intended to include all types of cancerous growths or tumorigenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness. Examples include, but are not limited to, solid tumors, hematologic cancers, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies, e.g., sarcomas and carcinomas of various organ systems (including adenocarcinomas and squamous cell carcinomas), such as those affecting the liver, lungs, breast, lymph, gastrointestinal tract (e.g., colon), genito-urinary tract (e.g., kidney, bungson epithelium), prostate, and pharynx. Adenocarcinoma includes malignant tumors such as most of colon cancer, rectal cancer, stomach cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma among lung cancer, small intestine cancer and esophageal cancer. Squamous cell carcinoma includes malignant tumors, such as in the lung, esophagus, skin, head and neck regions, mouth, anus, and cervix. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions described herein.
Non-limiting examples of cancers that can be suitably treated using the ADCs and combinations disclosed herein include gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). In addition, refractory or recurrent malignancies can be treated with the antibody molecules described in the present invention. Cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, cancer of the bladder, bone cancer, brain and CNS cancer, primary CNS lymphoma, Central Nervous System (CNS) tumor, breast cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, cancer of the connective tissue, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, renal cancer, laryngeal cancer, leukemias (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic or acute leukemia), liver cancer, lung cancer (e.g., small cell and non-small cell cancers), lymphomas including Hodgkin's and non-Hodgkin's lymphomas, lymphocytic lymphomas, melanomas such as malignant melanoma on the skin or in the eye, myelomas, neuroblastoma, oral cancer (e.g., lip cancer, cervical, Tongue, oral cavity cancer); ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, sarcoma: skin cancer, stomach cancer, thyroid cancer, uterine cancer, urinary cancer, liver cancer, cancer of the anal region, carcinoma of the fallopian tubes, vaginal cancer, cancer of the vulva, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, tumors of the vertebral axis, glioma of the brain stem, pituitary adenoma, kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, and other cancers and sarcomas and combinations of said cancers.
The present invention is further described below with reference to examples, which are not intended to limit the scope of the present invention. The experimental method of the present invention, in which the specific conditions are not specified, is usually performed according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.
Example 1: construction of cell lines highly expressing claudin 18.1 and 18.2(CLDN18.1 and CLDN18.2)
The human CLDN18.1, human CLDN18.2, mouse CLDN18.1 and mouse CLDN18.2 high expression cell strains used by the invention are completed by a stable cell strain construction platform of a company. The method comprises the following specific steps:
on day 1 of the experiment, 293T cells (cell bank Cat # GNHu17 of China Committee for type culture Collection of national academy of sciences) were seeded into two 6cm dishes, and the number of cells in each dish reached 7.5X 105And (4) respectively. On day 2, 4. mu.g each of the encapsulated plasmid (Biovector such as pGag-pol, pVSV-G, pBabe, plasmid vector bacterial cell Collection) and the plasmid pBabe-CLDN18.2 or pBabe-CLDN18.1 cloned with human or murine CLDN18.2 or CLDN18.1 gene was added to OPTI-MEM (Thermofish Scientific Cat #31985070) to give a final volume of 200. mu.l, and 200. mu.l of OPTI-MEM was prepared, and 36. mu.l of transfection reagent ctifen (Cat # F210, Shanghai culture Biotech Co., Ltd.) was added thereto, mixed, left at room temperature for 5min, and the mixture (200. mu.l each) was added dropwise to the cultured 293T cells. On day 3, the 293T cell culture solution was changed to 4ml of DMEM high-sugar medium (Shanghai Yuanbei Biotech Co., Ltd./Yuanbei: Cat # SEQ ID NO:320 KJ). CHO-K1 cells (cell bank Cat # SCSP-507 of China academy of sciences type culture Collection) were seeded on a 10cm dish at day 4 to reach a cell count of 5X 105. 293T cell supernatant (virus) was collected on day 5 and filtered through a 0.45 μm filter to obtain cultured cells10 μ g/ml polybrene (assist in Shanghai san Francisco Ltd., Cat #40804ES76) was added to CHO-K1 cells, mixed, placed in an incubator, and changed to DMEM/F1210% FBS medium (source organism, Cat # L310KJ) after 3-4 hours. CHO-K1 cells were passaged on day 7, and selection was carried out by starting the addition of puromycin at 10. mu.g/ml to the cells passaged on day 8 (source culture, Cat # S250J 0). And (3) dying the cells in a large amount in 2-3 days, replacing the culture medium to continue culturing until the cells are not dead any more, amplifying the cells in a large amount, screening the monoclonal cell strains, performing amplification culture, and freezing and storing the seeds.
The cell strains which stably express CLDN18 and are constructed by the invention are respectively marked as: human CLDN18.1+ cells (hcldn18.1+ cells), human CLDN18.2+ cells (hcldn18.2+ cells), murine CLDN18.1+ cells (mcldn18.1+ cells), and murine CLDN18.2+ cells (mcldn18.2+ cells). The protein sequences used were derived from publicly published databases, and the amino acid sequences of each protein are as follows.
Human CLDN18.1(hcldn 18.1):
>NP_057453.1,claudin-18isoform 1precursor[Homo sapiens]
MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV(SEQ ID NO:1)
human CLDN18.2(hcldn 18.2):
>NP_001002026.1claudin-18isoform 2[Homo sapiens]
MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV(SEQ ID NO:2)
murine CLDN18.1(mcldn 18.1):
>NP_062789.1claudin-18isoform A1.1precursor
MATTTCQVVGLLLSLLGLAGCIAATGMDMWSTQDLYDNPVTAVFQYEGLWRSCVQQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGVIGILVSIFALKCIRIGSMDDSAKAKMTLTSGILFIISGICAIIGVSVFANMLVTNFWMSTANMYSGMGGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLTPDDSNFKAVSYHASGQNVAYRPGGFKASTGFGSNTRNKKIYDGGARTEDDEQSHPTKYDYV(SEQ ID NO:3)
murine CLDN18.2(mcldn 18.1):
>NP_001181850.1claudin-18isoform A2.1[Mus musculus]
MSVTACQGLGFVVSLIGFAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGVIGILVSIFALKCIRIGSMDDSAKAKMTLTSGILFIISGICAIIGVSVFANMLVTNFWMSTANMYSGMGGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLTPDDSNFKAVSYHASGQNVAYRPGGFKASTGFGSNTRNKKIYDGGARTEDDEQSHPTKYDYV(SEQ ID NO:4)
example 2: anti-CLDN 18.2 antibody and binding (ELISA) assay for CLDN18.2+ and CLDN18.1+ cell lines
The cells obtained in example 1 were expanded to 5X 10 cells of a monoclonal cell line highly expressing human CLDN18.1, human CLDN18.2, murine CLDN18.1 or murine CLDN18.24After the plate was spread on a 96-well plate per well and incubated overnight in an incubator at 37 ℃, the supernatant was removed and fixed with an immunostaining fixative (Shanghai Biyuntian Biotechnology Co., Ltd. Cat # P0098) at 100. mu.l/well for half an hour at room temperature. After one wash with PBS (source culture organism, Cat # B320), 5% milk was blocked for 2 hours at 37 ℃ and washed 3 times with PBST. The sample to be tested (human or murine antibody, Jackson Immuno Research) is added. Incubate 1hr at 37 ℃ followed by PBST 3 washes. Adding Anti-human or mouse HRP 1: 250050 μ l/well, incubating at 37 deg.C for 1hr, washing with PBST for 3 times, developing color with TMB (Surmodic Cat # TTMB-1000-01), adding 50 μ l/well 1M H2SO4The reaction was terminated. Microplate reader (MultiskanGO Thermo model 51119200) reading, Graphpad prism5 for data analysis.
Example 3: cloning, expression and purification of recombinant proteins and antibodies
Cloning, expression and purification of the recombinant proteins/antibodies used in the present invention are performed according to molecular cloning methods well known to those skilled in the art.
Specifically, the expression vector used in the invention is purchased from Changsha Youbao biological science and technology limited company, and then EcoRI enzyme cutting sites (GAATTC) are introduced by Shanghai Jianxin biological medicine science and technology limited company (Jianxin organisms), so that the cloning of exogenous genes by a double-enzyme cutting or homologous recombination method is facilitated. Gene synthesis is carried out by a company such as Biotechnology engineering (Shanghai) Ltd. 293 cells, CHO-K were purchased from the cell bank of the culture Collection of the national academy of sciences.
The recombinant protein and the antibody are obtained by transient transfection expression and purification of 293 cells. Specifically, 293 cells were expanded in Gibco FreeStyle 293Expression Medium (Gibco, Cat # 12338018). Adjusting the cell concentration to 6-8X 10 before the start of transient transformation5cell/ml, 1% FBS (Aus Gene X FBS Excellent supplier: AusGeneX, China, Cat # FBSSA500-S), 8% CO at 37 ℃2Shake culturing for 24h, and microscopic examining the survival rate>95% and the cell concentration is 1.2X 106cell/ml。
300ml of the culture system cells were prepared, 15ml of Opti-MEM (Gibco, Cat #31985070) was dissolved in 150. mu.g each of the heavy and light chain plasmids, and 0.22 μm was subjected to filtration sterilization. Then, 15ml of Opti-MEM was dissolved in 600. mu.l of 1mg/ml PEI (Polysciences, Inc, Cat #23966-2) and allowed to stand for 5 min. In a500 ml culture system, 25ml of Opti-MEM (Gibco, Cat #31985070) was dissolved in 250. mu.g each of the heavy and light chain plasmids, and sterilized by 0.22 μm filtration. Then, 25ml of Opti-MEM was dissolved in 1000. mu.l of 1mg/ml PEI, and the mixture was allowed to stand for 5 min. Slowly adding PEI into the plasmid, incubating at room temperature for 10min, slowly dropping the mixed solution of the plasmid PEI into the culture flask while shaking, and adding 8% CO at 37 deg.C2Shaking culture for 5 days, collecting sample, and collecting supernatant at 3300G for 10min for purification.
And (3) purification: the samples were centrifuged at high speed to remove impurities and the column (biologies, Cat # F506606-0001) containing Protein A (Magselect, GEHealthcare Life Science, Cat #71-5020-91AE) was equilibrated with PBS pH7.4, washed 2-5 column volumes. The sample was run through the column. The column was washed with 5-10 column volumes of PBS (Producer organism, Cat # B548117-0500). Eluting the target protein with acetic acid of pH 3.50.1M, adjusting to neutral with Tris-HCl of pH 8.0, measuring the concentration with enzyme labeling instrument, packaging, and storing.
The recombinant human CLDN18.2(claudin18.2) extracellular region (D-70 at position 20 is fragment A) and Fc fusion protein of the invention were purified after transient transfection by 293 system. The protein can be used for detecting the serum titer of an immune mouse.
The antibody human CLDN18.2(anti-hcldn18.2) antibody (referred to as a control molecule or a positive molecule) was used as a comparison in the experiments of the present invention. The antibody is abbreviated as Ref (reference) in the invention, and the sequence is derived from WO 2014146672.
The anti-hCLDNN 18.2 antibody variable region sequence discovered by the invention is obtained from a monoclonal cell obtained by optimizing and screening hybridoma, and the optimized variable region sequence and different light and heavy chain constant regions are obtained by optimizing and screening humanized and patent drug sequences, and different antibody molecules are obtained by recombinant expression by the method and are used for detection and evaluation of activity, function and the like.
Example 4: discovery of anti-human CLDN18.2 antibodies
The anti-human CLDN18.2 monoclonal antibody of the present invention is obtained by immunizing a mouse with the human CLDN18.2 highly expressed cell strain (hcldn18.2+ cell) obtained in example 1, taking the spleen of the immunized mouse, performing hybridoma fusion, and screening and optimizing several million hybridoma clones.
Experimental mice, female, 4 weeks old (SJL from Beijing Wittingle laboratory animal technology, Inc., animal production license number: SCXK (Jing) 2016-. After the mice are purchased, the mice are raised in a laboratory environment for 1 week, and the dark period of the white day/night is adjusted to 20-25 ℃; the humidity is 40-60%. The mice were divided into 3 mice/group/cage.
Human CLDN 18.2-highly expressed cell lines (hcldn18.2+ cells, human CLDN18.2+ cells) constructed in example 1 were cultured, washed with DMEM medium (source culture organism, Cat # L310KJ) after trypsinization, and then resuspended in DMEM medium. At a ratio of 100. mu.l/1X 107Cells/mouse, i.p. immunized mice. At the time of first immunization, cells were mixed and immunized with Titermax (Sigma-Aldrich, T2684) at a ratio of 1: 1. After subsequent immunization for 1 week, 10 times, the serum titers of immunized mice were measured by plating human CLDN18.1+ cells and human CLDN18.2+ cells simultaneously using the ELISA method of example 2 or by plating human CLDN18.2 extracellular (ECL1) protein recombinantly expressed as in example 3 above, and the serum titers (titers) of mice were calculated using the ELISA values of the human CLDN18.1+ cell plating as a background. After 12-15 immunizationsMice with high serum titers and titers at plateau were selected for splenocyte fusion at 200. mu.l/2X 10 before fusion7Cell/mouse, 3 days after the mouse is washed, taking spleen lymphocyte and myeloma cell Sp2/0 cell of the mouse (CRL-8287TM) The hybridoma cells were plated in 96-well plates.
Hybridoma cell supernatants from 96-well plates were plated with human CLDN18.1+ cells and human CLDN18.2+ cells to detect antibody binding by hybridoma cells. Table 1a shows the results of the detection of a portion of the hybridoma supernatants.
TABLE 1a hybridoma fusion clones and detection of binding Activity for human CLDN18.2+ cell, human CLDN18.1+ cell
Since human CLDN18.2 and CLDN18.1 have up to 92% homology (240/261) and the protein is a transmembrane protein, only a small fraction of the peptide stretch is extracellular (e.g., 51 amino acids ECL1), immunogenicity is extremely low and the probability of producing specific antibodies is very small. Therefore, not only few hybridomas secreting CLDN 18-recognizing hybridomas were obtained in the above screen, but also in few hybridomas, the majority of antibodies in the hybridoma supernatant were antibodies that bound both human CLDN18.2 and CLDN 18.1.
Very surprisingly, the present inventors have found a hybridoma clone that secretes a supernatant that binds only to human CLDN18.2+ cells and does not bind to human CLDN18.1+ cells, see mab5, clone No. C13C1 in table 1 a. The data in Table 1a show that under the same screening conditions, the clone supernatant only bound to human CLDN18.2+ cells, which was found to be 1.41, while it hardly bound to human CLDN18.1+ cells, which had a binding activity reading of only 0.09.
Further, it was confirmed that the hybridoma cell line C13C1 unexpectedly found in the present invention can secrete a unique anti-human CLDN18.2 antibody. The C13C1 hybridoma cells were subjected to multiple limiting dilutions, and the monoclonal antibody after each dilution was carefully and finely screened, and finally, monoclonal cell strains secreting unique antibodies against human CLDN18.2 were found, and the results are shown in table 1 b.
TABLE 1b hybridoma monoclonal cell lines from hybridoma C13C1 optimized screening
As can be seen from the results in Table 1b, the monoclonal cell lines C13C1F1D3G6 and C13C1F1D3H5 secreted antibodies of the primary hybridoma clone C13C1 obtained by fine and optimized screening of the present invention retained binding to human CLDN18.2 cells, and read at 0.8895 and 0.8778, respectively. And no binding to human CLDN18.1 cells, reading 0.0859 and 0.0756, respectively. This reading is close to 0.081 of the present ELISA background. The initial hybridoma clone F2A4 also yielded monoclonal cell lines F2A4F6F3E4 and F2A4F6F3H7 by simultaneous selection. These monoclonal cell lines had the same binding activity as expected for human CLDN18.2 cells and human CLDN18.1 cells, and the data are shown in table 1 b. These results indicate that the present inventors found that monoclonal cell strains, such as C13C1F1D3G6, were capable of secreting unique antibodies that, surprisingly, only bind to human CLDN18.2, but not to human CLDN 18.1.
This means that the antibodies unexpectedly found by the present invention can effectively recognize only human CLDN18.2 protein, and have potential as monoclonal antibodies for treating tumors, particularly for treating cancer patients with overexpression of human CLDN18.2 protein, including but not limited to pancreatic cancer, gastric cancer, esophageal cancer, lung cancer, etc. Because the protein does not bind to human CLDN18.1 at all, it is expected that the toxic and side effects caused by the non-specific binding of therapeutic antibodies to proteins such as human CLDN18.1 can be avoided.
Example 5: the invention relates to the screening and identification of murine anti-human CLDN18.2 antibody
The murine antibody mab5b sequence of the present invention was obtained by extracting the antibody sequence secreted by the hybridoma monoclonal cell line C13C1F1D3G6 (table 1b) obtained in the above examples. The process of extracting antibody sequences from the preferred monoclonal cell lines of hybridomas is well known and commonly used by those skilled in the art.
Specifically, the present invention recovered 1X10 cells of the hybridoma monoclonal cell C13C1F1D3G6 found in the above example by expansion culture6Cells were treated with Trizol (Invitrogen, 15596-. And (3) carrying out PCR amplification by using cDNA obtained by reverse transcription as a template, and sequencing an amplification product to obtain an antibody light and heavy chain variable region sequence of mab5 b. The primers used are described in the handbook published by Novagen (TB326Rev. C0308).
The light chain nucleotide sequence of the anti-human CLDN18.2(anti-hCLDN18.2) monoclonal antibody obtained from the hybridoma monoclonal cell strain C13C1F1D3G6 is as follows, wherein the segment is a variable region sequence:
taatgggcttcaagatgaagtcacagtttctggtcctcatgtccctgctgttctgggtatctggtacctgtggggacattgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagttgca agtccagtcagagtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcc tcctaaactgttgatctactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgga acacatttcactctcaccatcagcagtgtgcaggctgaagacctggcagtttattactgtcagaatgattattttt atccattcacgttcggctcggggacaaagttggaaaaaaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttctgaacaactctaccccaaagaccatccatgccc(SEQ ID NO:5)
the anti-human CLDN18.2(anti-hCLDN18.2) monoclonal antibody heavy chain nucleotide sequence obtained from the hybridoma monoclonal cell strain C13C1F1D3G6 preferred by the invention is as follows, wherein the segment marked by the nucleotide sequence is variable region sequence:
taatgggatggaccgggatctttatctttctcctgtcagtaactgcaggtgttcactcccaggtccag ctgcagcagtctggagctgagctgataggacctgggacttcagtgaaggtgtcctgcaaggcctctggatacgcct tcagtaattacttgatagaatgggtaaaacagaggcctgaacagggccttgagtggattggtttgattaatcctgg aagtggtggcactaactacaatgagaagttcaagggcaaggcaacactgactgcagacaaatcctccagcactgcc tacatgcaactcagcagcctgacatctgatgactctgcggtctacttctgtgcaagggtctactatggtaactcct ttgcttactggggccaagggactctggtcactgtctctgcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctattaccgagcaagaaatgtcg(SEQ ID NO:6)
the amino acid sequence of the light chain variable region of the mab5b of the murine anti-human CLDN18.2(anti-hCLDN18.2) monoclonal antibody extracted from the hybridoma monoclonal cell strain discovered by the invention obtained by translation of the light chain base sequence is as follows:
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTHFTLTISSVQAEDLAVYYCQNDYFYPFTFGSGTKLEKK(SEQ ID NO:7)
the amino acid sequence of the heavy chain variable region of the murine anti-human CLDN18.2(anti-hCLDN18.2) monoclonal antibody mab5b extracted from the hybridoma monoclonal cell strain discovered by the invention obtained from the heavy chain base sequence is as follows:
QVQLQQSGAELIGPGTSVKVSCKASGYAFSNYLIEWVKQRPEQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARVYYGNSFAYWGQGTLVTVSA(SEQ ID NO:8)
the antibody mab5b extracted from the hybridoma monoclonal cell line discovered by the present invention was cloned (shown below in sequence) and expressed recombinantly in the light and heavy chain variable regions and constant regions of the antibody by the method described in example 3, and after purification, the binding activity to human hcldnn 18.1, hcldnn 18.2, murine mcldnn 18.1 and mcldnn 18.2 was detected simultaneously with the control antibody Ref, as shown in table 2a, table 2b and fig. 1 below.
The anti-hCLDN18.2 antibody mab5b light Chain (L Chain) of the invention:
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTHFTLTISSVQAEDLAVYYCQNDYFYPFTFGSGTKLEKKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:9)
the anti-hCLDN18.2 antibody mab5b heavy Chain (H Chain) of the invention:
QVQLQQSGAELIGPGTSVKVSCKASGYAFSNYLIEWVKQRPEQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARVYYGNSFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:10)
TABLE 2a binding Activity of the anti-hCLDN18.2 murine antibody mab5b of the invention with human CLDN18+ cell
TABLE 2b binding Activity of the anti-hCLDN18.2 murine antibody of the invention mab5b and murine CLDN18+ cell
Table 2a, fig. 1a shows that the anti-hcldn18.2 murine antibody mab5b and the control antibody (Ref) found in the present invention do not bind to hcldn18.1+ cell, no binding activity (ND) is detected by EC50 even at high concentration of 200nM, and the binding value Emax (which is the binding value at which binding plateaued at increased sample concentration, i.e., the maximum specific binding value) is still the background value. And both antibodies have good binding activity with hLDN 18.2+ cell. Surprisingly, the antibody mab5b of the invention binds more than 1-fold better than Ref (0.115 nM vs 0.249nM EC 50). More surprisingly, the maximum binding value Emax that can be achieved by mab5b is more than 36% (1.92-1.41)/1.41) higher than Ref.
Table 2b, fig. 1b show that the anti-hcldn18.2 murine antibody mab5b and the control antibody (Ref) found in the present invention do not bind to murine CLDN18.1+ cell, no binding (ND) is detected by EC50, no binding activity is detected even at high concentrations of 200nM, and the binding value Emax is still a background value. Both antibodies had good binding activity to murine CLDN18.2+ cells. Surprisingly, the antibody mab5b of the present invention bound more than 4-fold better than Ref (EC50 of 0.182nMvs 1.04 nM). More surprisingly, mab5b can achieve a maximum binding value Emax that is more than 1 times higher than Ref [2.21vs 1.0 ].
The above results indicate that the binding activity (EC50 and Emax) of the novel molecule mab5b found in the present invention is better than that of the control molecule. And the polypeptide has no binding activity with hLDN 18.1 and mLDN 18.1, which shows that mab5b not only has better binding activity, but also has excellent specificity. This indicates that mab5b provides the advantage of better efficacy and safety for the development of tumor therapy products. And also has better combination with the murine CLDN18.2, thus providing more convenient non-primate choice for preclinical research on mice.
Example 6: humanization of antibody mab5b of the invention
The activity of the antibody mab5b discovered by the invention is superior to that of Ref, and the antibody can be used for developing tumor treatment drugs. In order to reduce the risk in aspects such as immunogenicity in the process of drug development, for example, humanization is completed by a murine antibody, and the molecular characteristics after humanization are optimized so as to be convenient for drug development, the invention performs humanization screening on mab5b and sequence optimization work. The specific process is described below.
CDR definitions of antibodies there are a number of different methods in the art and these labeling CDR methods can be summarized in Table 3 below.
Table 3 summary of different methods for CDR definition of antibodies in the art
More information can be referred to the website: http:// www.bioinf.org.uk/abs/# cdrdef
The murine anti-human CLDN18.2 antibody mab5b obtained in example 5 above was labeled/annotated with CDR sequences according to the various definitions in table 3.
TABLE 4 anti-human CLDN18.2(anti-hCLDN18.2) antibody mab5b of the invention defines the CDR sequences according to CCG
TABLE 5 CDR sequences of the anti-human antibodies of the invention as defined by Kabat
TABLE 6 CDR sequences of the antibodies of the invention defined by AbM
TABLE 7 antibodies of the invention define CDR sequences according to Chothia
TABLE 8 CDR sequences of the antibodies of the invention defined by Contact
For the CDR analysis of the anti-hCLDNN 18.2 antibody (mab5b), based on the CDR regions of the light and heavy chains of the antibody identified by the markers according to the antibody marker system (as above), the variable region sequences of the light and heavy chains of the murine antibody mab5b are respectively compared with the human antibody germline database (v-base), so as to find out the germline of the light and heavy chains of the human antibody with high homology, and on the basis, computer modeling is carried out, the sites which may influence the antigen binding in the antibody structure are simulated, the key sites and combinations of back mutation are simulated, and the humanized antibody molecules with preferable activity are screened out.
Specifically, the human antibody germline which is compared with the mab5b light chain homology by sequence homology analysis was found to comprise IGKV4-1 × 01(F), IGKV2-28 × 01(F), IGKV2D-28 × 01(F), IGKV1-27 × 01(F), IGKV1-39 × 01(F), IGKV1D-39 × 01(F), IGKV2-40 × 01(F), IGKV2D-29 × 01(F), IGKV2D-40 × 01(F), and IGKV3-15 × 01 (F). Further comparison and analysis, human antibody germline light chain IGKV4-1 x 01(F) is preferred. Specifically, the CDR2 sequence of the selected human germline light chain IGKV4-1 × 01(F) is WASTRES, identical to the murine antibody mab5b light chain CDR2 sequence found in the present invention. The sequence alignment shows that the J gene region of the mab5b light chain has high homology with human antibody germline hJK1, hJK2.1, hJK2.2, hJK2.3 and hJK2.4, and further comparison and analysis show that hJK2.1 is preferably used for the J region of the mab5b light chain humanized human antibody germline to carry out humanized design, screening and sequence optimization.
The human antibody germline which is compared with the mab5b heavy chain homology by sequence homology comparison analysis is found to comprise IGHV1-69 x 02(F), IGHV1-69 x 06(F), IGHV1-69 x 08(F), IGHV1-69 x 09(F), IGHV1-69 x10 (F), IGHV1-69 x 04(F), IGHV1-69 x 14(F), IGHV1/OR15-2 x 02(P), IGHV1-69 x 01(F), IGHV1-69 x 11(F), and further comparison and analysis, preferably human germline heavy chain IGHV1-69 x 01(F) sequences are used for humanization of the antibody of the invention. The sequence alignment shows that the heavy chain J gene region of mab5b has high homology with human antibody germline heavy chain J genes hJh4.1, hJh4.2, hJh4.3, hJh1, hJh2, hJh3.1 and hJh3.2, and further comparison and analysis show that hJh4.1 is preferably used for humanized design, screening and sequence optimization of the human antibody germline J region of the mab5b heavy chain humanized antibody of the invention.
The CDR regions (see above for CDR definition) of the antibody mab5b of the present invention were grafted onto the selected humanized light and heavy chain human antibody germline templates and recombined with the IgG light and heavy chain constant regions. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, residues which directly interact with the CDR region, and residues which have important influence on the conformation of VL and VH are subjected to back mutation, the mutations and mutation combinations are screened, the influence on the activity of the antibody is observed, and the chemically unstable amino acid residues in the CDR region are optimized, so that an antibody molecule sequence with optimized structure, activity and the like, namely the humanized series optimized antibody molecule of the anti-human CLDN18.2 murine antibody mab5b, is obtained.
Specifically, in the analysis of the light chain of the antibody mab5b of the present invention, it was found that the light chain contains the sequences of CDR1 (positions L24-L34, underlined below) and CDR2 (positions L50-L56, underlined below) and and the present inventors have preferred the method of humanizationThe human germline light chain IGKV4-1 x 01(F) has high sequence homology. Among them, L24-L34CDR1 differ only by 5 amino acids, which are positions L29, L30A, L30C, L30E and L34, respectively, as shown in Table 9a below. The L50-L56 CDR2 are identical (see Table 9b below). The invention firstly carries out back mutation on five sites (L29, L30A, L30C, L30E and L34) of mab5b CDR1 to obtain amino acids at corresponding sites of human germline IGKV4-1 x 01(F), and the combined design is as shown in the following table 9 a.
TABLE 9a murine anti-hCLDNN 18.2 antibody mab5b sequence of the present invention amino acids at positions L24-L34 (CDR1, SEQ ID NO:11) as defined by Kabat and humanized
TABLE 9b amino acids at positions L50-L56 (CDR2) of mab5b sequence of murine anti-hCLDN18.2 antibody of the present invention
Amino acid position numbering | 50 | 51 | L52 | L53 | L54 | L55 | L56 |
mab5b CDR2 | W | A | S | T | R | E | S |
IGKV4-1*01(F)CDR2 | W | A | S | T | R | E | S |
The humanized design molecules Var1, Var2, Var3, Var4, Var5, Var6, Var7, Var8 and mab5b were cloned as described in example 3, and tested for human CLDN18.2+ cell binding activity as described in example 2, and the results are shown in Table 10 below.
TABLE 10 humanization optimization of the light chain CDR1 sequences of the antibodies of the invention
The above results indicate that the light chain CDR1 humanized optimized sequence KSSQS of the antibody mab5b of the present inventionVLNSGNQKNYLT(Var1,SEQ ID NO:70),KSSQSVLNSGNQKNYLA(Var2,SEQ ID NO:71),KSSQSLLNSGNNKNYLA(Var3,SEQ ID NO:12),KSSQSLLYSSNQKNYLT(Var4,SEQ ID NO:72),KSSQSLLYSSNQKNYLA(Var5,SEQ ID NO:73),KSSQSLLNSGNQKNYLA(Var8, SEQ ID NO:74), all retained the same (close) binding activity as mab5 b.
Furthermore, the humanized design of mab5b by the above-mentioned humanized method preferably results in the humanized light chain variable region of the mab5b antibody of the present invention having the following sequence:
L14:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:29)
L11:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTHFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:30)
L12:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:31)
L13:
DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:32)
L15:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTHFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:33)
the preferred sequences of the humanized heavy chain variable regions of the present invention obtained by the methods described above are as follows:
H51:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS(SEQ ID NO:34)
H52:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSTSTAYMELSSLRSEDTAVYFCARVYYGNSFAYWGQGTLVTVSS(SEQ ID NO:35)
H53:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS(SEQ ID NO:36)
H54:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS(SEQ ID NO:37)
the light chain variable region sequence described above, comprising any of the sequences listed as L14, L11, L12, L13, L15 and not listed, in combination with a kappa-type or lambda-type light chain constant region of a human antibody light chain, gives the humanized antibody light chain sequence of the present invention. The heavy chain variable region sequence comprises listed heavy chain variable region sequences such as H51, H52, H53, L54 and those not listed, and the constant region sequences of different subtypes such as hIgG1, 2, 3, 4 and the like which are combined to obtain the heavy chain sequence of the antibody. The light chain and the heavy chain are combined randomly to obtain the humanized antibody of the invention, and the sequence of the partially humanized antibody is shown in the following table 11.
TABLE 11 partially preferred sequences of the humanized antibodies of the present invention
Humanized Ab10 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:38)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39)
humanized Ab7 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTHFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:40)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39)
humanized Ab 8antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:38)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSTSTAYMELSSLRSEDTAVYFCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:41)
humanized Ab9 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTHFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:40)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSTSTAYMELSSLRSEDTAVYFCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:41)
humanized Ab6 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:42)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39)
humanized Ab11 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:42)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:43)
humanized Ab12 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:42)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:44)
humanized Ab13 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:45)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:43)
humanized Ab14 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:45)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:44)
humanized Ab15 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:45)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39)
the humanized antibody was cloned, expressed and purified by the method of example 3 of the present invention, and the binding activity of the humanized antibody to hLDN 18.2+ cell, hLDN 18.1+ cell was assayed and screened by the ELISA method of example 2, and the results are shown in Table 12 below and FIG. 2 a.
TABLE 12 binding Activity of the humanized anti-hCLDNN 18.2 antibody of the present invention with hCLDNN 18.2+ cell, hCLDNN 18.1+ cell
NA: not applicable; ND: is not detected
The results in Table 12 show that the antibody mab5b found in the present invention retains the advantage of higher binding activity of the murine antibody than the control antibody Ref after humanization, and that the EC50 of the humanized antibody Ab10 is 2 times better than Ref (0.117vs 0.345). Moreover, the binding maxima Emax of these humanised optimised molecules are 39.8% to 54.1% higher than for the control antibody Ref. Is better than the murine antibody mab5b than the control antibody.
In order to further optimize the humanized molecule of the present invention, the final sequence is preferably as consistent as possible with the germline light and heavy chains of the human antibody, so as to reduce the immunogenicity possibly caused by a small amount of sequences in the murine antibody, and preferably, a series of humanized antibodies are designed by the humanized optimized light chain CDR1 sequence Var3 (see Table 9), and specific binding activity screening is carried out. The results are shown in table 13 below and fig. 2 b.
TABLE 13 binding Activity of the humanized anti-hLDN 18.2 antibody of the present invention with hLDN 18.2+ cell, hLDN 18.1+ cell
ND: is not detected
The above results show that the humanized antibodies further optimized by the present invention (Table 13 and FIG. 2b) have different numbers of amino acids in the back mutation, including Ab1-Ab20 (Table 11). Of these, there are 6 back-mutations, such as Ab10 (light chain 1, heavy chain 5); there are 6 back mutations and the light chain CDR1 is humanizedly optimized, such as Ab6 (light chain 1, heavy chain 5); there were 2 back mutations such as Ab14 (0 light chain, 2 heavy chains); there were only 1 back-mutation such as Ab11 (1 light chain, 0 heavy chain), and no back-mutation at all, such as Ab 13. These optimized humanized molecular activities, including EC50 and Emax activities, all maintained the same level as Ab10 (the already optimized mab5b humanized molecule in table 12) and all did not bind to hcldn18.1+ cells.
The Ab13 antibody molecule was found particularly unexpectedly to be free of any back mutations, i.e. to be a fully humanized antibody molecule, and the CDR1 sequence was optimized for humanization, with the same binding activity (EC50 and Emax) as Ab6, Ab 10.
The above results show that the antibody molecule obtained by humanization and optimized screening of the sequence of the murine antibody mab5b of the present invention comprises humanized FR regions only, and the light chain CDR1 retains the wild type (no mutation), such as Ab 10; or in addition to the FR region, the light chain CDR1 has also been humanized and optimized sequence Var3, and the obtained Ab6, Ab11-15 and the like all maintain the binding activity and are superior to that of a control molecule, the EC50 is 1 time stronger than that of the control molecule, the Emax is 30-50% higher than that of the control molecule, and the combination of the two is different from that of hLDN 18.1+ cells.
Example 7: optimization of antibody sequence deamidation (deamidation) sensitive site sequence
Analysis of possible sites, particularly post-transcriptional modification (PTMs) sites of CDR regions, including aggregation, deamidation sensitivity (asparaginization, sites (NG, NS, NH, etc.), aspartate isomerization (DG, DP) sensitive sites, N-glycosylation (N- { P } S/T) sensitive sites, oxidation sensitive sites, etc., by computer structure modeling analysis of the mab5b sequence of the invention and humanized optimized sequences (Table 9a, 9b, 11 above) of the invention, revealed that the antibody light Chain CDR1(CDR1, L Chain) L30A, L30B NS, heavy Chain CDR3(CDR3, H Chain) H99, H100 NS, wherein L30A, H99 para (N) may be sensitive to degradation of the antibody when used in drug-related formulations, for the L30A, L30B (NS) of the light chain CDR1 of the antibody of the present invention; the H99 and H100 (NS) points of the heavy chain CDR3 are mutated, and the preferred scheme is as follows:
TABLE 14 optimized design of deamidation sensitive site sequences of the antibody sequences of the present invention
*: NA, not applicable (not changed)
The preferred deamidation sensitive site-optimized variable region sequences described above are as follows:
L20:
DIVMTQSPDSLAVSLGERATINCKSSQSLLTSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:46)
L21:
DIVMTQSPDSLAVSLGERATINCKSSQSLLNTGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:47)
L22:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNTGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:48)
L23:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNTGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:49)
H60:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGTSFAYWGQGTLVTVSS(SEQ ID NO:50)
H61:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSS(SEQ ID NO:51)
H62:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSS(SEQ ID NO:52)
H63:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSTSTAYMELSSLRSEDTAVYFCARVYYGNTFAYWGQGTLVTVSS(SEQ ID NO:53)
H64:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSS(SEQ ID NO:54)
and (3) expressing the antibody by combining different light chains and heavy chains through the optimized sequence after the optimized deamidation sensitive site is optimally designed, and then further screening the binding activity. The antibody combinations are described in part in the following table.
TABLE 15 deamidation sensitive site optimized antibodies of the invention
*: -represents no mutation
Ab30 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:38)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:55)
ab34 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:42)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:55)
ab35 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNTGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:56)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:57)
ab36 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNTGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:58)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:57)
the above-mentioned preferred antibodies were expressed and purified according to example 3, and then tested for binding activity to human CLDN18.2+ cells according to example 2, and the results are shown in tables 16a and 16b, Table 16c and FIGS. 3a and 3 b.
TABLE 16a deamidation sensitive site optimization of antibody Activity by the humanized anti-hCLDNN 18.2 antibody of the present invention
*: ab14 as a control sample for this assay (without NT mutation)
TABLE 16b deamidation sensitive site optimization of antibody Activity by the humanized anti-hCLDNN 18.2 antibody of the present invention
*: ab14 as a control sample for this assay (without NT mutation)
TABLE 16c deamidation sensitive site optimization of antibody Activity by the humanized anti-hCLDNN 18.2 antibody of the present invention
The results in Table 16a show that Ab21 (light chain CDR1NS- > TS and heavy chain CDR3NS- > TS) "has little" binding activity (>16 nM). Ab23 (light chain CDR1NS- > NT, heavy chain CDR3NS- > TS) also had "little" binding activity (>40 nM). Ab22 (light chain CDR1NS- > TS, heavy chain CDR3NS- > NT) had reduced binding activity but did not completely disappear (EC50 ═ 1.37nMvs Ab14EC50 ═ 0.35). That is, although N- > T at position L30A of light chain CDR1, since N at position H99 of heavy chain CDR3 was not mutated, the binding activity of the antibody returned from "almost nothing" to 1.37nM, which was reduced by nearly 3-fold compared to normal Ab 14. The binding activity of Ab56 (light chain CDR1NS- > NT, heavy chain CDR3NS- > NT) is the same as that of normal molecule Ab14, with EC50 of 0.295 nM.
Taken together the results in Table 16a indicate that N at position L30A of the light chain CDR1 and N at position H99 of the heavy chain CDR3 of the antibody of the invention are important for binding by the antibody of the invention, and that mutations at these points (e.g., to T) directly result in loss of activity or complete lack of activity. However, the mutation of S at position L30B of the light chain and S at position H100 of the CDR3 of the heavy chain to T had no effect on the binding activity.
The data in Table 16b further demonstrate that the S mutation at position L30B of the light chain alone to T or the S mutation at position H100 of the heavy chain CDR3 alone to T had no effect on the binding activity.
Very unexpectedly, the data in Table 16b indicate that Ab30 (heavy chain CDR3, NS->NT) complete loss of binding activity (EC50)>11nM, i.e., the binding curve has been practically identical to that of the negative control, and no specific binding to antigen has occurred), but Ab34 contains the same heavy chain (CDR3, NS->NT) without loss of activity. This indicates that the loss of Ab30 activity was not due to CDR3, NS->The change in NT. The only difference in these two molecules was the difference in the light chain CDR1, i.e., Ab30 light chain is L14 and its CDR1 is KSSQSLLNSGNQKNYLT(ii) a Ab34 light chain is L12, CDR1KSSQSLLNSGNNKNYLAThe underlined parts are the differences of the two CDR1 sequences. This unexpected finding indicates that when the heavy chain CDR3 has an H100S mutation to a T (to avoid potential deamidation), the light chain CDR1 sequence must be KSSQSLLNSGNNKNYLA(i.e., CDR1 sequence optimized Var3, see Table 9 a). Light chain CDR1 if KSSQSLLNSGNQKNYLTIf (underlined is the difference in the sequences of the two CDRs 1), the entire anti-molecule will lose binding activity completely (see Ab30 in FIG. 3 a). This is oneIt was found that the antibody CDR1 of the invention could not be mutated at the H100 th position S of the heavy chain if the L30E position was Q and the L34 position was T (underlined), e.g. to avoid potential deamidation of the S at H100 position to T, otherwise the whole antibody lost binding activity.
To further confirm the light chain CDR1 sequence KSSQSLLNSGNQKNYL of the antibodies of the present inventionT(Q at position L30E and T at position L34) and humanised optimised CDR1 sequence KSSQSLLNSGNNKNYLA(N at position L30E and A at position L34) on NS mutations in the light chain CDR1 and heavy chain CDR3 of the antibodies of the invention (to avoid potential deamidation), we compared Ab10(CDR 1: KSSQSLLNSGN)QKNYLT) And Ab6(CDR 1: KSSQSLLNSGNNKNYLA) The only difference between these two molecules is their light chain CDR1 (the underlined amino acids are the differences between the two). And simultaneous NT-treatment on the light chain CDR1 and heavy chain CDR3 of the two molecules>NS mutation, and two obtained Ab35 and Ab36 antibodies. Table 16c results show that Ab35 lost binding activity completely (EC50)>62nM) (see FIG. 3b, Ab 35).
This data confirms the light chain CDR1 sequence KSSQSLLNSGN discovered by the present inventionNKNYLAN at position L30E and a at position L34 (underlined), i.e. the CDR1 humanised optimised sequence did not affect the light chain CDR 1S at position L30B (italics), and or the heavy chain H100S was mutated to avoid potential deamidation (e.g. S at position L30B to T or/and S at position H100 to T).
If the CDR1 sequence KSSQSLLNSGNQKNYLTQ at position L30E and T at position L34 (underlined) neither the S at position L30B (italics) nor the S at position H100 of the CDR1 (for example, the S at position L30B is mutated to T or the S at position H100 is mutated to T) can be mutated, and any such mutation would abolish the binding activity of the antibody.
The above results are combined to show that the light chain CDR1L30A of the antibody of the invention, NS position 30B of L30; the NS at the H100 position of the heavy chain CDR3H99 can pass through NS->The NT was optimized by mutation to reduce the risk of deamidation, but only N at position L30E and A at position L34 of CDR1 (i.e., sequence KSSQSLLNSGN for humanization optimization)NKNYLA) Then, if the sequence of the region is KSSQSLLNSGNQKNYLTThe antibody is completely inactivated (the underlined parts are sequence differences of the two).
Example 8: assay for Activity of antibody Fc sequence (variant) of the present invention
The antibody variable region of the present invention and the different light and heavy chain constant regions of human antibodies, including but not limited to the combination of different light chains (kappa, lambda type light chains, etc.) and heavy chain constant regions (hIgG2, hIgG4, hIgG1) of human antibodies as listed in example 3, especially human IgG1Fc sequence variants, such as different forms of DEL or EEM at positions 356-358, can be obtained as different antibody variant forms. Table 17 lists some of the antibodies of the invention and variant forms of the Fc sequences, including the Fc region sequences at positions 356-358 of DEL or EEM.
TABLE 17 antibodies of the invention differing in the light and heavy chain constant regions
Numbering | Light chain | Heavy chain | hIgG1, 356-358 position |
Ab10 | L14 | H51 | DEL |
Ab42 | L14 | H51 | EEM |
Ab6 | L12 | H51 | DEL |
Ab43 | L12 | H51 | EEM |
Ab13 | L13 | H53 | DEL |
Ab48 | L13 | H53 | EEM |
Ab51 | L21 | H64 | DEL |
Ab52 | L21 | H64 | EEM |
Ab14 | L13 | H54 | DEL |
Ab53 | L13 | H54 | EEM |
Ab24 | L21 | H61 | DEL |
Ab56 | L21 | H61 | EEM |
The partial antibody sequences are as follows:
ab42 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:38)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:59)
ab43 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:42)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:59)
ab24 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATINCKSSQSLLNTGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:60)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:61)
ab51 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATINCKSSQSLLNTGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:60)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:62)
ab56 antibody amino acid sequence:
light chain:
DIVMTQSPDSLAVSLGERATINCKSSQSLLNTGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:60)
heavy chain:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:63)
the above-mentioned preferred antibodies were expressed and purified according to the method of example 3, and then tested for binding activity to human CLDN18.2+ cells according to the method of example 2, and the representative data are shown in table 18 below. The results indicate that the above changes in the light and heavy chain constant regions, including DEL or EEM at positions 356-358 of hIgG1, respectively, do not affect the activity of the antibodies of the invention.
TABLE 18 binding Activity of the humanized anti-hCLDN18.2 antibody IgG1Fc 356-358 DEL or EEM of the present invention
Example 9: sequence optimization of the Fc region (human IgG1) of the antibody of the present invention for ADCC and CDC activities
One of the action mechanisms of the antibody specifically combined with human CLDN18.2 for treating tumors is that the antibody can achieve the purpose of treating tumors by killing tumor cells through Fc-mediated effector cells (effector cells). The human Fc region (hIgG1Fc) of the antibody mediates ADCC and CDC effects of effector cells, can specifically enhance the effect of targeting tumor cells, and causes side effects outside targets for non-specific targets. There have been many studies on ADCC and CDC mediated by the Fc region of human antibodies (hIgG1 Fc). The present inventors confirmed the effector effects (ADCC and CDC) of the Fc region of the found antibody molecule against human blood cells. Specifically, the human antibody Fc region (hIgG1Fc) of the antibody of the present invention was subjected to different mutations, and ADCC and CDC activities of these mutants were evaluated, and the mutation design and activity data are shown in tables 19 and 20a and 20b, respectively.
TABLE 19 active site design of the antibody Fc (IgG1) of the present invention for ADCC and CDC
After the antibody is obtained by expression and purification of the above-mentioned preferred antibody by the method of example 3, the activity of the antibody after optimization of the Fc sequence is detected by ADCC (antibody-dependent cytotoxicity assay) and CDC (complement-dependent cytotoxicity assay), respectively. In particular, the amount of the solvent to be used,
ADCC:
the hLDN 18.2+ cells constructed in example 1 were cultured normally. The culture medium was DMEM/F12 supplemented with 10% FBS (Cat # L310KJ, Shanghai-culture Biotech Co., Ltd.) as target cells for ADCC in this experiment.
One day before the experiment, cultured hCLDNN 18.2+ cells were taken, and 5000 cells/well were plated in 96-well plates. On the day of the experiment, PBMC cells (isolated from human peripheral blood of the present invention, donated from volunteers of the same company) were prepared, and PBMC were suspended at a concentration of 150000 cells/50. mu.l in serum-free RPMI1640 medium (culture organism, Cat # L210 KJ). The test drug was prepared in serum-free RPMI1640 and diluted 3-fold at an initial concentration of 40. mu.g/ml.
The cultured target cells (hLDN 18.2+ cells) were removed, the supernatant carefully aspirated off, and the prepared PBMC was added at 50. mu.l/well; simultaneously adding 50 mul/hole prepared samples to be detected with different concentrations, then placing the target cells at 37 ℃,5%CO2the incubation was carried out in an incubator for 4 hours and LDH was detected.
The LDH Kit is cytotoxin LDH Assay Kit-WST, which is purchased from Dong ren chemical technology (Shanghai) Co., Ltd., product number CK 12. The operation method is carried out according to the instruction, the pore plate is taken out, 100 mu l of Workingsolution is added into each pore, aluminum foil is wrapped and protected from light, the reaction is carried out for 10-40min at room temperature, multiskanGO (ThermoFisher) enzyme labeling instrument 490nM reads, the detection is carried out once every 10min, the proper reaction time data is taken, and the data is analyzed and processed by Graphpadprism 5.
CDC:
The hLDN 18.2+ cells constructed in example 1 were used as target cells for CDC in this experiment. hCLDN18.2+ cells were cultured normally in DMEM/F12 plus 10% FBS (with ADCC). One day before experiment, collecting target cells, counting, preparing 1 × 105Cells/ml, 100. mu.l/well were added to 96-well cell culture plates. 37 ℃ and 5% CO2Incubate overnight for future use.
On the day of the experiment, the medium in the 96-well plate cells was removed and washed 2 times with PBS for use. The antibody to be tested was diluted with serum-free medium (RPMI1640) at an initial antibody concentration of 20. mu.g/ml, 5-fold. 50 μ l/well diluted antibody was added to PBS washed target cell culture plates (0 μ g/ml antibody wells with 100 μ l/well of fresh medium as control wells) with 6 duplicate wells for each concentration point. 37 ℃ and 5% CO2Incubate for 15 min.
Preparation of complement: fresh serum was taken into a sterilized centrifuge tube. Half of the serum was incubated in a 56 ℃ water bath for 30 minutes to inactivate complement as a negative control. Inactivated and non-inactivated sera were made up into sera with RPMI1640 medium, respectively: RPMI1640 medium 40%: 60%, i.e. 40% serum, 60% RPMI 1640.
Adding 50 mul/hole diluted serum into a target cell culture plate containing antibodies to be detected with different concentrations, wherein the final concentration of the serum is 20%; the initial concentration of sample (antibody) was 10. mu.g/ml. Complement serum was added to the first 3 wells and complement inactivating serum was added to the last 3 wells. Incubate at 37 ℃ in a 5% CO2 incubator for 2 hours, and then remove for detection with an LDH kit.
The LDH Kit is cytotoxin LDH Assay Kit-WST, which is purchased from Dong ren chemical technology (Shanghai) Co., Ltd., product number CK 12. The operation method is carried out according to the instruction, the pore plate is taken out, 100 mu l of Workingsolution is added into each pore, aluminum foil is wrapped and protected from light, the reaction is carried out at room temperature, the detection is carried out once every 10min, the proper reaction time data is taken, the MultiskanGO (ThermoFisher) microplate reader 490nM reading is carried out, and Graphpadprism5 analyzes and processes the data.
TABLE 20a assay of ADCC Activity of the antibody Fc (IgG1) mutants of the invention
Sample (I) | EC50(μg/ml) |
Ab6 | 1.40 |
Ab59 | Undetectable (No binding) |
Ab60 | 0.431 |
Neg IgG* | Undetectable (No binding) |
Neg IgG: non-specific antibodies not binding to the target
TABLE 20b CDC Activity assay of antibody Fc (IgG1) mutants of the present invention
Sample (I) | EC50(μg/ml) |
Ab6 | 0.290 |
Ab59 | Undetectable (No binding) |
Ab60 | 0.973 |
Ab24 | 0.255 |
Ab65 | Undetectable (No binding) |
Ab66 | 0.927 |
Neg IgG* | Undetectable (No binding) |
Neg IgG: non-specific antibodies not binding to the target
The above results indicate that if the antibody of the present invention is in the form of hIgG1, the F243 position of the Fc region is mutated, for example, F243L, to completely lose ADCC and CDC activities of the antibody of the present invention; if the Fc region has combined mutations at the 239, 330 and 332 positions, such as S239D/A330L/I332E, the CDC activity of the antibody of the invention is reduced.
Example 10: evaluation of ADCC and CDC activities of different humanized molecules of the antibody
In order to evaluate the ADCC and CDC activities of the humanized antibody molecules of the present invention, the ADCC and CDC activities of the humanized antibody molecules of the present invention were measured using the same method as the above-described examples, together with the control molecule (Ref), and the results are shown in tables 21a, 21b and fig. 4 below.
TABLE 21a ADCC Activity evaluation of different humanized molecules of antibodies of the invention
Sample (I) | EC50(μg/ml) |
Ab10 | 1.55 |
Ab6 | 1.40 |
Ref | 1.39 |
Neg IgG* | Undetectable (No binding) |
Neg IgG: non-specific antibodies not binding to the target
TABLE 21b CDC Activity evaluation of different humanized molecules of the antibodies of the invention
Sample (I) | EC50(μg/ml) |
Ab10 | 0.648 |
Ab35 | Undetectable (No binding) |
Ab6 | 0.293 |
Ab36 | 1.75 |
Ab14 | 0.265 |
Ab24 | 1.30 |
Ab13 | 0.374 |
Ab51 | 2.12 |
Ref | 2.31 |
Neg IgG* | Undetectable (No binding) |
Neg IgG: non-specific antibodies not binding to the target
The above results indicate that the ADCC activity of the humanized antibody of the present invention is equivalent to that of the control antibody (Ref) (Table 21 a). Unexpectedly, the CDC activities of the humanized molecules of the present invention, including humanized molecules containing different back mutations, were close (0.293 μ g/ml, 0.374 μ g/ml, 0.265 μ g/ml for EC50) and 1-fold or more better than Ab10(0.648 μ g/ml), and especially unexpectedly the CDC activities of Ab6, Ab13, Ab14, etc. were nearly 10-fold better than the control antibody (2.31 μ g/ml for EC50) (see table 21b, fig. 4). The CDC activities of Ab36 and Ab24 were also better than those of the control antibody Ref.
Ab35(CDR1, CDR3NS mutant, see example 7, table 16c) was undetectable for CDC activity due to loss of binding activity.
Example 11: evaluation of apoptosis Activity of antibody-induced tumor cells (hCLDN18.2+ cells) of the present invention
In order to detect the effect of the antibody, particularly the preferred humanized antibody, on inducing the apoptosis of hLDN 18.2+ cells (tumor cells), we used the hLDN 18.2+ cells constructed in the embodiment 1 of the present invention to detect the apoptosis activity of the tumor cells induced by the antibody. hCLDNN 18.2+ cells were cultured normally (DMEM/F12 with 10% FBS in the medium, supplier: Shanghai culture Biotech Co., Ltd., product number: L310) as the cells used in this experiment. At the beginning of the experiment, hCLDNN 18.2+ cells were plated in 96-well plates at a density of 2X 104A hole. Adherence was achieved by culturing overnight at 37 ℃ in 5% CO 2. Antibody sample preparation: serum-free DMEM/F12 medium was used to prepare 0. mu.g/ml, 1. mu.g/ml, 3. mu.g/ml, 10. mu.g/ml antibody samples. The overnight anchorage-cultured hCLDNN 18.2+ cells were removed, the medium was discarded, and the cells were washed twice with PBS. Prepared antibody samples were added at different concentrations, 100. mu.l/well, respectively. After further incubation for 24h, LDH was detected.
The LDH Kit is cytotoxin LDH Assay Kit-WST, which is purchased from Dong ren chemical technology (Shanghai) Co., Ltd., product number CK 12. The operation method is carried out according to the instruction, the pore plate is taken out, 100 mu l of Workingsolution is added into each pore, the light is protected by methods of wrapping aluminum foil and the like, the reaction is carried out at room temperature, 490nM reading of Multiskan GO (ThermoFisher) microplate reader is carried out at different time points (10min, 20min, 30min, 40min and 50min), the optimal reaction time is found out, and the reading value is analyzed and processed by Graphpad prism 5.
The results are shown in FIGS. 5a and 5 b. The results in FIGS. 5a and 5b show that the activity of the humanized antibodies Ab10, Ab6, Ab14, Ab24, Ab36 and the like of the present invention in inducing apoptosis in tumor cells is better than that of the control molecule (Ref), and the activity is better/stronger by 3-10 times, and the concentration of these molecules at 10. mu.g/ml is already equivalent to that of the control molecule at 30. mu.g/ml or even stronger than that of the positive molecule at 30. mu.g/ml.
Specifically, Ab 610. mu.g/ml increased the tumor cell apoptosis activity (46.7%) by more than 2 times than the positive control at the same concentration (15.3%); even more than 1 time better than the positive control 30 mug/ml (19.6%); whereas the activity of inducing apoptosis of tumor cells at a concentration of 30. mu.g/ml (85.1%) was more than 3 times better than that of the control antibody at the same concentration (19.6%).
More surprisingly, Ab 610 μ g/ml increased tumor apoptosis activity (46.7%) by 41% over the same concentration of Ab10 (33.2%); the activity of inducing apoptosis of tumor cells at 30. mu.g/ml (85.1%) was more than 1-fold stronger than that of Ab10 (34.7%) at the same concentration.
Example 12: the antibody of the invention inhibits the proliferation of tumor cells (hCLDN18.2+ cells)
For the purpose of examining the inhibition of tumor cell proliferation by the antibody of the present invention, activity assay was carried out using hCLDNN 18.2+ cells constructed in example 1. Specifically, hCLDNN 18.2+ cells were cultured normally (medium: DMEM/F12 containing 10% FBS, supplier: Shanghai-source culture Biotech Co., Ltd., cat # L310). At the beginning of the experiment, hLDN 18.2+ cells growing in logarithmic phase were plated in 96-well plates at a density of 3X 103A hole. The cells were cultured overnight in 5% CO2 adherent culture at 37 ℃. Antibody sample preparation: antibody samples of 1. mu.g/ml, 10. mu.g/ml, 30. mu.g/ml were prepared with DMEM/F12 (source culture) containing 10% FBS. The hCLDNN 18.2+ cells cultured by the overnight adherence are taken out, the culture medium is discarded, the cells are washed once by PBS, and then prepared antibody samples with different concentrations are respectively added in 100 mul/well. After further culturing for 72h, the cells were detected with CCK-8 kit.
The CCK-8 Kit is Cell Counting Kit-8 available from Dongzren chemical technology (Shanghai) Co., Ltd., product number CK 04. The protocol was followed by taking out the 96-well plate, adding 10. mu.l CCK-8 solution to each well (taking care not to generate bubbles in the wells, otherwise the reading would be affected), incubating the plate in the incubator for 1-4h to find the optimal detection time point, Multiskan GO (ThermoFisher) microplate reader reading at 450nM, and processing the data with Graphpad prism5 analysis. The results are shown in Table 22 below.
TABLE 22 inhibition of tumor cell (hCLDNN 18.2+ cell) proliferation activity (% inhibition) by the preferred humanized antibodies of the invention
Sample/concentration | 1μg/ml | 10μg/ml | 30μg/ |
Neg IgG | |||
0 | 0.34 | 0.1 | |
Ab10 | 2.17 | 2.93 | 3.16 |
Ab6 | 3.29 | 6.12 | 6.2 |
Ref | 2.34 | 2.94 | 3.9 |
The results in Table 22 show that the negative antibody at a concentration of 1. mu.g/ml, 10. mu.g/ml, or 30. mu.g/ml inhibits the activity of tumor cells by less than 1%, i.e., the background level. Ab10 with the concentration of 1 mu g/ml, 10 mu g/ml and 30 mu g/ml inhibits the activity (inhibition rate) of tumor cells at 2.17-3.16 percent, which is close to the inhibition rate of Ref at 2.34-3.9 percent. Ab6 inhibits tumor cell activity much stronger than Ref, for example, the inhibition rate of 10. mu.g/ml is 6.12% higher than 2 times of Ref (2.94%).
Example 13: detection of binding Activity of the humanized antibody of the present invention and murine CLDN18
Binding activity of the humanized preferred antibody of the present invention to murine CLDN18.1+ cell and murine CLDN18.2+ cell was examined according to the method of the previous example 2. During the screening process, we screened for a clone (antibody L180) that binds to both human and murine CLDN 18.1. As a control for the assay, L180 and mcldn18.1+ cell binding activity EC50 was 0.48nM, indicating that the murine CLDN18.1+ cell constructed according to the present invention specifically binds to the anti-CLDN 18.1 antibody. Whereas, the preferred humanized antibody of the invention did not bind to mcldn18.1+ cell, and both retained the same binding activity as murine antibody mab5b to mcldn18.2+ cell, as shown in table 23 below.
TABLE 23 binding Activity of preferred humanized antibodies of the invention with mCLDn18.2+ cells
Sample (I) | EC50(nM) | Emax |
mab5b | 0.375 | 2.17 |
Ab10 | 0.371 | 2.27 |
Ab35 | ND# | ND |
Ab6 | 0.594 | 2.47 |
Ab36 | 0.518 | 2.18 |
Ab14 | 0.399 | 2.39 |
Ab24 | 0.574 | 2.22 |
Ab13 | 0.422 | 2.37 |
Ab51 | 0.474 | 1.91 |
Neg IgG* | Without bonding | 0.19 (background) |
#: not detected. Neg IgG: non-specific antibodies not binding to the target
The above results indicate that the preferred humanized antibodies of the invention retain binding activity to mcldn18.2+ cell. EC50 and mab5b before humanization (EC50 ═ 0.375nM in the same assay) had the same binding activity. Emax is between 1.91(Ab51) -2.47 (Ab6), and close to mab5b (2.17).
Example 14: evaluation of in vivo pharmacodynamic Activity of antibody of the present invention
In order to evaluate the antitumor activity of the antibody of the present invention, in vivo efficacy evaluation of the antibody of the present invention was carried out by subcutaneously implanting BALB/c nude mice with an animal efficacy model established by hCLND18.2+ cells (constructed in example 1) or a gastric cancer cell line NUGC4 (Shanghai SulBiotech, Ltd.).
Specifically, the hCLDNN 18.2+ cell culture medium was DMEM/F12 (source culture organism) plus 10% fetal bovine serum (Shanghai Boliter Biotech Co., Ltd., cat # BS-0002-. The NUGC4 cell culture medium was RPMI1640 (source culture organism) supplemented with 10% fetal bovine serum. The culture conditions were 37 ℃ and 5% CO2. BALB/c Nude mice, female, 4-week-old, with a weight of 18-20g, purchased from Shanghai West Pulbiki laboratory animals Co., Ltd (production license number: SCXK (Jing) 2012-0001), at room temperature of 20-25 deg.C, with a humidity of 40-60%, fed freely with water, and bred adaptively for 3-4 days. The padding and the cleaning cage are replaced at proper time. Cells in the logarithmic growth phase were collected and counted.
For hCLDNN 18.2+ cell allotransplantation model, hCLDNN 18.2+ cells were washed 2 times with PBS and resuspended to 1X10 cells8And/ml. Mice were inoculated subcutaneously on the left flank with 0.1ml of 1X10 in total7Cell/cell. Tumors were selected to grow to a volume of about 120 and 180mm3The mice were divided into groups of 5-6 mice each.
For the stomach cancer NUGC4 cell allograft model, NUGC4 cells are washed 2 times with RPMI1640, and Matrigel is added to the cells in a ratio of 1:1, resuspending the cells in the mixture, 1X108And/ml. Mice were inoculated subcutaneously on the left flank with 0.1ml of 1X10 in total7Cell/cell. Tumors were selected to grow to a volume of about 150-3The mice were divided into groups of 5-6 mice each.
And preparing a sample to be detected by PBS and sterilizing. Blank is a PBS no sample control, and an antibody unrelated to the target, i.e., a negative antibody (Neg IgG) control. Intraperitoneal injection, 200ug/100 μ l/tube. 2 times/week for several weeks. The day of sample injection was day 0. Body weight, tumor volume were measured before each dose and data were recorded.
Tumor size calculation formula:
tumor volume TV (mm)3) 0.5 × (tumor major diameter × tumor minor diameter)2)
Relative tumor growth rate (T/C%) (T-T0)/(C-C0)%)
Tumor inhibition rate (TGI) ═ 1-T/C100%
Wherein T0, T is the tumor volume at the beginning and end of the experiment of the sample group; c0, C is the tumor volume at the beginning and end of the control experiment, respectively.
The results of the in vivo drug effect in animals are shown in fig. 6a, 6b (hcldn18.2+ cell xenograft model) and table 24 below (nudc 4 tumor cell xenograft model).
In a tumor allograft model established by using human CLDN18.2 high-expression cells (hCLDN18.2+ cells), the results of FIG. 6a show that the antibodies Ab10 and Ab6 of the invention have very good in-vivo pharmacodynamic activity as well as positive antibody molecules (Ref), and the inhibition of the growth of tumor cells and/or the killing of tumor cells (inhibition rate) reaches more than 90 percent; unexpectedly, the light chain CDR1 and heavy chain CDR3 deamination sensitive site optimized, light chain CDR1 humanized optimized preferred antibody Ab36 completely inhibited tumor growth with significantly better in vivo potency than Ab6, Ab10, and the control positive antibody (Ref). The results in FIG. 6b show that the fully humanized antibody (without back mutations) Ab13 of the present invention, and its light chain CDR1 and heavy chain CDR3 deamination sensitive site optimized fully humanized antibody Ab51, and light chain CDR1 and heavy chain CDR3 deamination sensitive site optimized humanized antibody Ab24 with only 2 back mutations in the heavy chain, all showed the same in vivo efficacy as the control antibody.
TABLE 24 evaluation of the efficacy of the antibodies of the invention in the gastric cancer cell line NUGC4 tumor model
NA-not adapted, i.e.blank control.
In the tumor model established by the human gastric cancer cell line NUGC4, the results (Table 24) show that the humanized and preferred antibodies Ab10 and Ab6 of the invention both show certain drug effects, the tumor inhibition rate is 10-20%, and the dosage dependence is shown. In the same model, the control antibody (Ref) had no tumor-inhibiting effect, as did the PBS control and negative antibody (Neg IgG). This result indicates that the antibody of the present invention has an in vivo efficacy superior to that of the positive control antibody.
Example 15: antibody mouse Pharmacokinetic (PK) evaluation of the invention
As described in example 13 above, the antibodies of the invention have good binding activity to mouse CLDN18.2, which provides preclinical non-primate species selection for the antibodies of the invention. The present invention evaluates the Pharmacokinetic (PK) profile of the antibodies of the invention in mice.
Specifically, experimental Balb/c mice, female, 6-week old, were purchased from Shanghai Sphall-BikKa laboratory animals Co., Ltd. After the mice are purchased, 6 mice are placed in each cage, and feed and water are obtained in an unlimited amount. Feeding in laboratory environment for 3 days at 20-25 deg.C; humidity 40-60%, 12/12 light/dark cycle adjustment. The day before the start of the experiment, the body weight of the mice was measured, and 20-25g of the mice were assigned to group numbers of 3 mice/group. On the day of the experiment, each mouse was subcutaneously injected with test drug Ab10 and control antibody (Ref) at a dose of 10mg/kg and 100 ul/mouse, respectively.
Orbital bleeds were performed 0, 1, 6, 24, 26, 30, 50, 55, 71, 79, 98, 143, 167, 191, 215, and 240 hours after injection of the mice, respectively. Centrifuging the blood sample, collecting supernatant, storing at-20 deg.C, and testing.
After the blood sample was collected, the blood concentration in the serum was measured by the ELISA method described in example 2. Before formal detection, one mouse serum is taken and subjected to gradient dilution, and the optimal dilution ratio of the serum is determined. All samples were tested by ELISA at the optimal dilution ratio and the results were analyzed using the calculation formula of T1/2 and EXCEL software, as shown in Table 25 below.
TABLE 25 PK evaluation of the antibodies of the invention in mice
The results in Table 25 above show that the antibody Ab10 of the invention has a Cmax 57% higher than the control antibody (Ref) ([407.7-259.3 ]/259.3). More surprisingly, the half-life T1/2 of the antibody Ab10 of the invention was more than 30.7 hours longer (170.1-139.4) than the control antibody (Ref), and the in vivo superiority of T1/2 in mice was up to 30 hours, which is expected to be greater in humans. These results indicate that the humanized antibody of the present invention has excellent PK properties, especially half-life T1/2, compared to the control antibody, and is expected to bring at least advantages of drug efficacy (long time) and cost (low administration frequency) for clinical use.
Example 16: antibody affinity assay of the invention (KinExA)
CLD18.2 is a cellular transmembrane protein, four times transmembrane, with both extracellular regions ECL1 and 2 being only 20-50 amino acids long. The antibodies of the invention specifically bind to the extracellular domain of CLDN 18.2. Biacore detection methods, which routinely express antigen (20-50 amino acids) in combination with antibodies, do not give a good estimate of the specific affinity of the antibodies of the invention for the extracellular domain of the target protein. Therefore, the present invention detects the affinity of the antibody and hcldn18.2+ cells using the KinExA method. The method is carried out according to the instructions of a KinExA4000 instrument, namely, the antibodies Ref and Ab10 to be detected are Constant Binding Partner (CBP) respectively, and hLDN 18.2+ cells are Titrant. Cells (Titrant) were diluted with a fixed concentration antibody (CBP) gradient, free antibody (free CBP) not bound to cells was captured by anti-human IgG Fc column after incubation, signal values were obtained with anti-human Fc Alexa Flour 647, and antibody affinity was calculated by KinExA self-contained software.
Specifically, to determine the concentration of the appropriate antibody dilution, a Signal test was first performed by estimating the appropriate concentration based on the estimated affinity, and it was determined that 500ul of 120pM Ref antibody and 100pM Ab10 antibody were used as Signal 100%, respectively, at which a satisfactory detection net Signal value was obtained, and PBS blank was used as a negative Signal value (NSB). The CBP concentrations were determined as the concentrations of 120pM Ref antibody and 100pM Ab10 antibody. In the following equilibration experiments, centrifugation at 300g for 10 minutes was carried outTwo tubes of hLDN 18.2+ cells were collected, the number of cells per tube was 5X 108One (positive rate 100% by FACS). The cells were washed once with PBS, centrifuged at 300g for 10 minutes, and collected in 15ml centrifuge tubes. 15ml of 120pM Ref and 100pMAb10 antibody solutions were prepared, respectively. To 5X 108120pM Ref antibody solution was added to 2ml of each cell, and the cells were diluted in a 2-fold gradient using 120pM Ref antibody solution as a buffer at an initial concentration of 2.5X 108Cells/ml, 18 gradients, 0.6ml each. To 5X 108100pM Ab10 antibody solution was added to 2ml of each cell, and the cells were diluted in a 2-fold gradient starting at a concentration of 2.5X 10 with 100pM Ab10 antibody solution as buffer8Cells/ml, 18 gradients, 0.6ml each. Cells were incubated with the suspension of antibody for 2h at room temperature with shaking. After the incubation, 450g was centrifuged for 10 minutes, and the supernatant was collected. A solution of 1. mu.g/ml anti human Fc AlexaFlour 647 was prepared. The samples were placed in the appropriate positions in the tube rack. Signal values were detected with a KinExA3200 instrument and affinity data were obtained. The results are shown in the following table. The results show that: ab10 has an affinity (13.3pM) that is more than 10 times higher than that of Ref antibodies.
TABLE 26 detection of affinity (M) of the antibodies of the invention
Sample (I) | Ab10 | Ref |
KD | 1.33×10-11 | 1.44×10-10 |
Example 17: detection of the endocytic Activity of the antibodies of the invention
hCLDNN 18.2+ cell grows toAt 90%, trypsinized and the cells resuspended in FACS buffer (PBS + 1% BSA) to a final concentration of 1X106Cells/ml. Add 500. mu.l of cell suspension to a 1.5ml centrifuge tube, add the fluorescently labeled antibody control antibody (antibody not bound to hLDN 18.2+ cell), control antibody Ref, test sample (humanized antibody is preferred in the present invention) Ab10, Ab6 (mix-n-stand CF488antibody labeling kit, Sigma-Aldrich, Cat # MX488S100-1kit or mix-n-stand CF633antibody labeling kit, Sigma-Aldrich, Cat # MX633S100-1kit labeling steps were performed according to the kit instructions), incubate 1 hour on ice at a final concentration of 1. mu.g/ml or 10. mu.g/ml, and wash three times with precooled buffer FACS. The sample was removed 1/5 and placed on ice as a binding value sample for direct flow detection. The remaining 4/5 cells were resuspended in 1640+ 10% FBS preheated at 37 ℃ and placed 1/4 directly on ice as a 0-hour endocytosis sample, the remaining cells were incubated in 37 ℃ incubator for 1hr, 2hr, and 3hr respectively, taken out, precooled on ice, terminated endocytosis, centrifuged at 1300rpm for 3 min at 4 ℃ and the supernatant discarded. Mu.l strip buffer (0.05M glycine, pH 2.45+0.1M NaCl) was added at 0hr, 1hr, 2hr, 3hr, centrifuged at room temperature for 7min at 1300rpm at 4 ℃ for 3 min, the supernatant was discarded, and washed once with FACS buffer. All samples were loaded with 150. mu.l of 4% paraformaldehyde (Biotechnology Cat # E672002), fixed at 4 ℃ for half an hour and tested on the machine (Beckman Cytoflex flow cytometer). The results are shown in the following table.
TABLE 27 endocytic Activity of the antibodies of the invention (FACS fluorescence intensity, CF 488-labeled antibody)
The above results (labeling of the test antibody with CF 488) show that at an antibody concentration of 1. mu.g/ml (left half of the table above), the binding fluorescence intensity of the control antibody (Ref) (1939) and the fluorescence intensity value of the negative antibody (antibody not bound to hLDN 18.2+ cells) (1747) are close, i.e., background values. Furthermore, fluorescence intensity values (streaking values) at 0h, 1h, 2h, and 3h were also close to background (1747). The binding values (fluorescence intensities) of the antibodies Ab10 and Ab6 of the invention are 27200 and 16300, respectively, which are 15 times and 9 times of background, and 14 times (27200/1939) and 8.4 times (16300/1939) of the control antibody. This further demonstrates that the binding Emax of the antibodies of the invention is stronger than the control antibody (Ref).
The fluorescence intensity value, the background value, of the negative antibody (antibody not bound to hLDN 18.2+ cells) when the antibody concentration was increased to 10. mu.g/ml (right half of the above table) was 1284-3485 (underlined number). The fluorescence intensity (binding value) of the control antibody (Ref) was 17900, which is 4-fold higher than background (3485), indicating that specific binding of the fluorescently labeled antibody was detected up to 10. mu.g/ml for Ref. In the same concentration, the binding strength (fluorescence value) of the antibodies Ab10 and Ab6 of the invention is 138000 and 86000 which are 39 times and 24 times of background (3485) and 7.7 times (138000/17900) and 4.8 times (86000/17900) of Ref. This further illustrates that the binding Emax of the antibodies of the invention is much stronger than the control antibody (Ref). This is also consistent with the above KinExA assay.
The data in the table above are expressed by percent (%) endocytosis (fluorescence intensity at the time point to be measured-0 h fluorescence intensity)/binding value according to the formula, and the data in the table below are obtained.
TABLE 28 endocytic Activity (% endocytosis) of the antibody of the present invention (CF 488-labeled)
Background: the reading value is the background value and no endocytosis exists; NA: not applicable, no endocytosis at this time point
The above results show that the preferred antibodies Ab10 and Ab6 of the invention show better endocytosis at a concentration of 1. mu.g/ml (left half part of the table), 1h, 2h and 3h, and the endocytosis at 3h is 28% and 25%, respectively. Under the same conditions, the Ref antibody was identical to the negative control antibody, and no endocytosis was observed.
When the amount of antibody was increased to 10. mu.g/ml (right half of the upper table), the control antibody still had very little endocytosis (7% -8%), almost near background, i.e., no endocytosis. The endocytosis of the antibodies Ab10 and Ab6 of the invention still increased with time to 3h, 23% and 18%, respectively. This is less than 28% and 25% of the 1. mu.g/ml antibody concentration, suggesting that 10. mu.g/ml is supersaturated for the antibody of the invention rather than the optimized antibody endocytosis concentration, since the antibody binding activity of the invention is much better than Ref.
These results indicate that the preferred humanized antibodies of the invention are endocytic antibodies. Whereas the control antibody (Ref) is not an endocytic antibody, or the endocytosis is very weak.
To further demonstrate the endocytic activity of the preferred antibodies of the invention, labeled antibodies and endocytic activity analysis were performed using a fluorescent dye CF633(Sigma-Aldrich, Cat # MX633S100-1kit) other than CF488, with the detection instrument being a BDFACS Calibur flow cytometer. The results are given in the table below.
TABLE 29 endocytic Activity of the antibody of the present invention (FACS fluorescence intensity, CF 633-labeled antibody)
The above results (labeling of test antibody with CF633) show that the fluorescence intensity value of no binding of negative antibody and hLDN 18.2+ cells, i.e., the background value, is 23 at an antibody concentration of 1. mu.g/ml (left half of the table above). Fluorescence intensity values (streaking values) at 0h, 1h, 2h and 3h are also close to background (12.7-13.7, namely, the background values are less than 100 within 2 times). The binding fluorescence intensity (62.5) of the control antibody (Ref) was close to background (negative antibody, 23), in the 2-fold range (62.5/23 ═ 2.7). Furthermore, the combined fluorescence intensity values at 0, 1, 2, 3h were all weak (15.5-33.4), and in particular the readings at 1, 2, 3h did not change (33.9, 33, 7, 33.4), indicating that these values are substantially close to background levels.
The binding values (fluorescence intensities) of the antibodies Ab10 and Ab6 of the invention were 854 and 690, respectively, 37 times (854/23) and 30 times (690/23) that of the background, and 14 times (854/62.5) and 11 times (690/62.5) that of the control antibody, respectively. This further demonstrates that the binding Emax of the antibodies of the invention is much stronger than the control antibody (Ref). This is also consistent with the above KinExA results.
The fluorescence intensity value, i.e. the background value, of the negative antibodies (antibodies that do not bind to hcldn18.2+ cells) when the antibody concentration was increased to 10 μ g/ml (right half of the table above) was 23.2-14.9 (underlined numbers). The fluorescence intensity (binding value) of the control antibody (Ref) was 198, 8.5 times that of the background (23.2), indicating that Ref was able to detect specific binding of the fluorescently labeled antibody up to 10. mu.g/ml. At the same concentration, the binding strength (fluorescence value) of the antibodies Ab10 and Ab6 of the invention is 3229 and 2237 respectively, 139 times and 96 times of background (23.2) and 16 times (3229/198) and 11 times (2237/198) of Ref respectively. This further illustrates that the binding Emax of the antibodies of the invention is much stronger (fluorescence readings more than 10-fold stronger) than the control antibody (Ref).
The data in the table above are expressed by percent (%) endocytosis (fluorescence intensity at the time point to be measured-0 h fluorescence intensity)/binding value according to the formula, and the data in the table below are obtained.
TABLE 30 endocytic Activity (% endocytosis) of the antibody of the present invention (CF 633-labeled)
Background: the reading value is the background value and no endocytosis exists; NA: not applicable, no endocytosis at this time point
The results show that the antibodies Ab10 and Ab6 preferably show better endocytosis in 1h, 2h and 3h when the concentration of the antibodies Ab10 and Ab6 is 1 mu g/ml, and the endocytosis in 3h is 35.1 percent and 30.4 percent respectively. Under the same conditions, the Ref antibody (the binding strength is within 2-3 times of the background) has no endocytosis as the negative control antibody.
When the amount of antibody was increased to 10. mu.g/ml (right half of the upper table), the control antibody still had very little endocytosis (5% -8%), almost near background, i.e., no endocytosis. The endocytosis of the antibodies Ab10 and Ab6 of the invention increased with time to 3h, which were 27.3% and 28.9%, respectively (see FIG. 7). This is less than 35.1% and 30.4% of the 1. mu.g/ml antibody concentration, suggesting that 10. mu.g/ml is already a supersaturated concentration for the antibody of the invention, rather than a concentration that optimizes endocytosis, because the antibody binding activity of the invention is much better than Ref.
The results of the experiments combining the two different fluorochromes (CF488 and CF633) show that the preferred humanized antibodies Ab10 and Ab6 of the invention are endocytic antibodies. This is completely different from the control antibody (Ref). The control antibody was not endocytosed, or endocytosis was very weak, near background levels.
Example 18: preparation of anti-CLDN 18.2 antibody Ab10 coupled toxin SMCC-DM1(ADC1)
The preparation method of the anti-CLDN 18.2 antibody Ab10 coupled toxin SMCC-DM1(ADC1) of the present invention refers to the methods disclosed in patents CN106188293A and US2009202536a1, specifically the following steps.
The ADC1 sample is detected and analyzed by an LC-MS method, and the obtained sample is proved to have no free toxin small molecules. The absorbance peaks of a252 and a280 were detected by a spectrophotometer (UV method), and the ratio DAR of the resulting ADC1 toxin to antibody was determined to be 4.4.
Example 19: preparation of anti-CLDN 18.2 antibody Ab10 coupled toxin MC-VC-PAB-MMAF (ADC2)
The preparation method of the anti-CLDN 18.2 antibody Ab10 coupled toxin MC-VC-PAB-MMAF (ADC2) of the present invention refers to the methods disclosed in patents CN106188293A and US20140127211a1, specifically the steps are as follows.
After 5ml of the above solution 1a was taken, 0.15ml of 2.0M hydroxylamine hydrochloride solution was added thereto, and the mixture was reacted at 25 ℃ for 30 minutes with shaking, the reaction mixture was desalted and purified by Sephadex G25 gel column (eluent phase: 0.05M PBS solution having pH 6.5), to obtain an intermediate 1b in the above line, i.e., Ab 10-propylthiol solution.
The resulting ADC2 was at a final concentration of 1.21mg/ml and was dispensed for storage at 4 ℃ until use. The ADC2 sample is detected and analyzed by an LC-MS method, and the obtained sample is proved to have no free toxin small molecules. The absorbance peaks of a252 and a280 were detected by a spectrophotometer (UV method), and the ratio DAR, i.e., y, of the resulting ADC2 toxin and antibody was determined to be 4.8.
Example 20: anti-CLDN 18.2 antibody Ab10 cytotoxic conjugate ADC1, ADC2 binding activity assay
The binding activity of the antibody Ab10 of the invention, as well as the Ab10 antibody cytotoxic conjugate ADC1, ADC2 and hLDN 18.2+ cel, was tested by ELISA as described in example 2, and the results are shown in Table 8 below and FIG. 8.
TABLE 31 cytotoxic conjugate of Ab10 antibody ADC1, ADC2 binding activity (EC50)
Sample (I) | ADC1 | ADC2 | Ab10 |
EC50(nM) | 0.344 | 0.310 | 0.164 |
The above results show that the binding activity (EC50) of Ab10 antibody cytotoxin conjugates ADC1, ADC2 and target cells (hcldn18.2+ cell) and antibody Ab10 are somewhat weaker than those of the former, but within a 2-fold (error) range, the binding curves are shown in fig. 8.
Example 21: anti-CLDN 18.2 antibody Ab10 cytotoxic conjugate ADC1, ADC2 endocytosis assay
The endocytic activity of the antibody Ab10 of the present invention and the Ab10 antibody cytotoxic conjugates ADC1 and ADC2 were detected by the same method as described above for the detection of the endocytic activity of the antibody of the present invention (example 17). On the basis of the above examples, the labeling method (using CF633 dye), the antibody concentration (2.5. mu.g/ml), and the time (1 hour) were optimized for endocytosis detection conditions, and the results are shown in the following table.
TABLE 32 cytotoxic conjugate ADC1 and ADC2 of Ab10 antibody of the invention for detection of endocytic activity
Sample (I) | Binding value1) | Before endocytosis (0h) | After endocytosis (1h) | Proportion of endocytosis (%)3) |
Negative antibodies | 932 2) | 935 | 944 | NA |
Ab10 | 6135 | 1191 | 2055 | 14.1 |
ADC1 | 4136 | 1200 | 2119 | 22.2 |
ADC2 | 4254 | 1788 | 2754 | 22.7 |
1) The binding value is the FACS detection reading value of the CF633 labeled antibody before endocytosis and after endocytosis;
2) underlined numbers are background values for the present FACS binding;
3) endocytosis ratio (1 hour) 100 (post-endocytosis-pre-endocytosis)/binding value; NA: not applicable to
The above results indicate that the Ab10 antibody cytotoxin conjugate ADC1, ADC2 of the present invention retained the endocytic activity of antibody Ab 10. That is, toxin-linked antibody Ab10 did not affect the endocytic activity of the antibody.
Example 22: anti-CLDN 18.2 antibody Ab10 cytotoxic conjugates ADC1, ADC2 inhibit target cell proliferative activity
To examine the cytotoxic conjugate ADC1 and ADC2 of Ab10 antibody against the target cell proliferation activity, the cell proliferation was examined by the CCK8 method (kit available from Donren chemical technology (Shanghai) Co., Ltd., cat. No. CK04. according to the instructions). Specifically, hcldn18.2+ cells (target cells) and hcldn18.1+ cells (non-CLDN 18.2 target cells, as control cells) were cultured in DMEM/F12 medium containing 10% FBS, 10ul cck8 was added to each well 2 hours before the end of 72 hours of culture, the culture was continued in an incubator for 2 hours, OD450nM readings were obtained with a Multiskan GO microplate reader, and the data were analyzed with Graphpadprism 5. Percent (%) inhibition of cell proliferation was 100 × (1- (OD450 sample/OD 450 control well)), the results are shown in the table below, fig. 9a, 9 b.
TABLE 33 cytotoxic Ab10 antibody conjugates of the invention ADC1 and ADC2 inhibit the proliferative activity of target cells (IC50, nM)
As a result, the Ab10 antibody cytotoxin conjugates ADC1 and ADC2 molecules of the present invention can significantly inhibit the proliferation of target-specific cells (hcldn18.2+ cell) with a dose-dependent curve IC50 of 11.2nM and 0.71nM, respectively (fig. 9a, fig. 9 b). ADC1 had no inhibitory effect on non-target cells (hcldn18.1+ cells not bound to Ab10 antibody used in this experiment) with a safety window as high as 3000. Inhibition of non-target specific cells was seen by ADC2 at very high concentrations (IC50 of 18.5nM), and the dose curve (fig. 9b) shows that this inhibition is due to non-targeted toxicity directly caused by high doses (up to 10-100 nM). This is consistent with the highly toxic nature of MMAF to which ADC 2is attached. The safety window of ADC2 reaches 26.
Example 23: anti-CLDN 18.2 antibody Ab10 cytotoxic conjugate targeted cytotoxic activity detection
To detect that the Ab10 antibody cytotoxic conjugate of the invention specifically targets CLDN18.2+ cytotoxicity, the present example uses ADC1 as an example to evaluate the target cytotoxic activity of the anti-CLDN 18.2 antibody Ab10 cytotoxic conjugate of the invention by detecting the release of LDH in the cell supernatant.
Specifically, hcldn18.2+ cells (target cells) and hcldn18.1+ cells (non-CLDN 18.2 target cells, as control cells) were cultured to logarithmic growth phase with DMEM/F12 medium containing 10% FBS. One day before the experiment, cultured hLDN 18.2+ cells and hLDN 18.1+ cells were collected, resuspended in DMEM/F12 medium containing 10% FBS, and the cell concentration was adjusted to 4X 104Cells/ml, 100. mu.l/well into 96-well cell culture plates; 37 ℃ and 5% CO2Incubating overnight; fruit of Chinese wolfberryOn the day of assay, the medium in the cells of the 96-well plate was removed and DMEM/F12 medium containing 2% FBS was added to each well for use; preparing a gradient diluted drug to be detected ADC1 by using a DMEM/F12 culture medium containing 2% FBS, and using a fresh culture medium for 0 mu g/ml antibody wells as control wells; adding prepared antibody samples with different concentrations into the cells, wherein each well contains 50 mu l of antibody samples, and each concentration is three wells; 37 ℃ and 5% CO2After incubation in the incubator for 72 hours, the supernatant was removed and assayed for release of LDH using an LDH kit (purchased from donnay chemical technology (shanghai) ltd., product No. CK 12). The detection method is carried out according to the instruction. The process data was analyzed using Multiskan GO microplate reader with 490nM readings, Graphpad prism 5. Percent (%) cell killing was 100 × (OD490 test sample-OD 490 control wells)/(OD 490 cells total lysis-OD 490 control wells). The results of the experiments are shown in the following table.
TABLE 34 evaluation of Ab10 antibody cytotoxic conjugate ADC1 Targeted cytotoxicity (EC50, nM) of the invention
The result shows that the Ab10 antibody cytotoxicity conjugate has strong specific targeting hLDN 18.2+ positive cytotoxicity, and EC50 is less than 1 nM. Whereas the cytotoxic effect against non-targeted cells (hcldn18.1+ cells not bound to Ab10 antibody used in this experiment) was weak (no cytotoxicity). In particular, the ratio of toxicity of ADC1 between targeting specific cells and non-specific cells (EC50) was as high as 156. This specificity window (safety window) shows that the non-targeted toxicity of the molecule is weak and the safety is reliable.
Example 24: in vivo efficacy of anti-CLDN 18.2 antibody Ab10 cytotoxic conjugate
The in vivo efficacy of the antibody Ab10 cytotoxic conjugate of the invention was evaluated using an animal model and experimental method of subcutaneous transplantation of hCLND18.2+ cells in BALB/c nude mice in the same manner as in example 14, using ADC1 as an example. The results are shown in Table 35.
TABLE 35 evaluation of the efficacy of the antibody conjugate drug ADC1 of the present invention in tumor models
NA-not adapted, i.e.blank control.
The results in table 35 show that the antibody toxin conjugate drug ADC1 of the present invention showed very good tumor suppression effect at low concentration (2mg/kg) and only 4 administrations, 2 weeks (14 days), 46% inhibition rate, and very significant difference from the control group (Pvalue < 0.001).
SEQUENCE LISTING
<110> Shanghai Jianxin biomedicine science and technology Co., Ltd
<120> drug conjugate of anti-CLDN 18.2 antibody, and preparation method and use thereof
<130>P180115840C
<160>74
<170>PatentIn version 3.5
<210>1
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Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu
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Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg
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Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
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Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
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Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
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Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
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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
<210>3
<211>264
<212>PRT
<213>Mus musculus
<400>3
Met Ala Thr Thr Thr Cys Gln Val Val Gly Leu Leu Leu Ser Leu 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 Ala Val Phe Gln Tyr Glu Gly
35 40 45
Leu Trp Arg Ser Cys Val Gln 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 Val Ile Gly Ile Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Asp Asp Ser
100 105 110
Ala Lys Ala Lys Met Thr Leu Thr Ser Gly Ile Leu Phe Ile Ile Ser
115 120 125
Gly Ile Cys Ala Ile Ile Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Ser Gly Met Gly Gly
145 150 155 160
Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala
165 170 175
Ala Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly
180 185 190
Val Met Met Cys Ile Ala Cys Arg Gly Leu Thr Pro Asp Asp Ser Asn
195 200 205
Phe Lys Ala Val Ser Tyr His Ala Ser Gly Gln Asn Val Ala Tyr Arg
210 215 220
Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Arg Asn
225 230 235 240
Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Asp Glu Gln Ser
245 250 255
His Pro Thr Lys Tyr Asp Tyr Val
260
<210>4
<211>264
<212>PRT
<213>Mus musculus
<400>4
Met Ser Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Phe 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 Val Ile Gly Ile Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Asp Asp Ser
100 105 110
Ala Lys Ala Lys Met Thr Leu Thr Ser Gly Ile Leu Phe Ile Ile Ser
115 120 125
Gly Ile Cys Ala Ile Ile Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Ser Gly Met Gly Gly
145 150 155 160
Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala
165 170 175
Ala Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly
180 185 190
Val Met Met Cys Ile Ala Cys Arg Gly Leu Thr Pro Asp Asp Ser Asn
195 200 205
Phe Lys Ala Val Ser Tyr His Ala Ser Gly Gln Asn Val Ala Tyr Arg
210 215 220
Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Arg Asn
225 230 235 240
Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Asp Glu Gln Ser
245 250 255
His Pro Thr Lys Tyr Asp Tyr Val
260
<210>5
<211>530
<212>DNA
<213>Mus musculus
<400>5
taatgggctt caagatgaag tcacagtttc tggtcctcat gtccctgctg ttctgggtat 60
ctggtacctg tggggacatt gtgatgacac agtctccatc ctccctgact gtgacagcag 120
gagagaaggt cactatgagt tgcaagtcca gtcagagtct gttaaacagt ggaaatcaaa 180
agaactactt gacctggtac cagcagaaac cagggcagcc tcctaaactg ttgatctact 240
gggcatccac tagggaatct ggggtccctg atcgcttcac aggcagtgga tctggaacac 300
atttcactct caccatcagc agtgtgcagg ctgaagacct ggcagtttat tactgtcaga 360
atgattattt ttatccattc acgttcggct cggggacaaa gttggaaaaa aaacgggctg 420
atgctgcacc aactgtatcc atcttcccac catccagtga gcagttaaca tctggaggtg 480
cctcagtcgt gtgcttctga acaactctac cccaaagacc atccatgccc 530
<210>6
<211>528
<212>DNA
<213>Mus musculus
<400>6
taatgggatg gaccgggatc tttatctttc tcctgtcagt aactgcaggt gttcactccc 60
aggtccagct gcagcagtct ggagctgagc tgataggacc tgggacttca gtgaaggtgt 120
cctgcaaggc ctctggatac gccttcagta attacttgat agaatgggta aaacagaggc 180
ctgaacaggg ccttgagtgg attggtttga ttaatcctgg aagtggtggc actaactaca 240
atgagaagtt caagggcaag gcaacactga ctgcagacaa atcctccagc actgcctaca 300
tgcaactcag cagcctgaca tctgatgact ctgcggtcta cttctgtgca agggtctact 360
atggtaactc ctttgcttac tggggccaag ggactctggt cactgtctct gcagccaaaa 420
cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact aactccatgg 480
tgaccctggg atgcctggtc aagggctatt accgagcaag aaatgtcg 528
<210>7
<211>113
<212>PRT
<213>Mus musculus
<400>7
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Lys
100 105110
Lys
<210>8
<211>118
<212>PRT
<213>Mus musculus
<400>8
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ile Gly Pro Gly Thr
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210>9
<211>220
<212>PRT
<213>Artificial Sequence
<220>
<223> mab5b light chain
<400>9
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Lys
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210>10
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> mab5b heavy chain
<400>10
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ile Gly Pro Gly Thr
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
3540 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>11
<211>17
<212>PRT
<213>Mus musculus
<400>11
Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu
1 5 10 15
Thr
<210>12
<211>17
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR1 humanized optimized sequence
<400>12
Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Asn Lys Asn Tyr Leu
1 5 10 15
Ala
<210>13
<211>7
<212>PRT
<213>Mus musculus
<400>13
Trp Ala Ser Thr Arg Glu Ser
1 5
<210>14
<211>9
<212>PRT
<213>Mus musculus
<400>14
Gln Asn Asp Tyr Phe Tyr Pro Phe Thr
1 5
<210>15
<211>10
<212>PRT
<213>Mus musculus
<400>15
Gly Tyr Ala Phe Ser Asn Tyr Leu Ile Glu
1 5 10
<210>16
<211>17
<212>PRT
<213>Mus musculus
<400>16
Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 1015
Gly
<210>17
<211>9
<212>PRT
<213>Mus musculus
<400>17
Val Tyr Tyr Gly Asn Ser Phe Ala Tyr
1 5
<210>18
<211>5
<212>PRT
<213>Mus musculus
<400>18
Asn Tyr Leu Ile Glu
1 5
<210>19
<211>10
<212>PRT
<213>Mus musculus
<400>19
Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn
1 5 10
<210>20
<211>7
<212>PRT
<213>Mus musculus
<400>20
Gly Tyr Ala Phe Ser Asn Tyr
1 5
<210>21
<211>6
<212>PRT
<213>Mus musculus
<400>21
Asn Pro Gly Ser Gly Gly
1 5
<210>22
<211>13
<212>PRT
<213>Mus musculus
<400>22
Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr
1 5 10
<210>23
<211>13
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR1 humanized optimized sequence
<400>23
Leu Asn Ser Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr
1 5 10
<210>24
<211>11
<212>PRT
<213>Mus musculus
<400>24
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
1 5 10
<210>25
<211>8
<212>PRT
<213>Mus musculus
<400>25
Gln Asn Asp Tyr Phe Tyr Pro Phe
1 5
<210>26
<211>6
<212>PRT
<213>Mus musculus
<400>26
Ser Asn Tyr Leu Ile Glu
1 5
<210>27
<211>13
<212>PRT
<213>Mus musculus
<400>27
Trp Ile Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn
1 5 10
<210>28
<211>10
<212>PRT
<213>Mus musculus
<400>28
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala
1 5 10
<210>29
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized light chain variable region L14
<400>29
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
15 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>30
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized light chain variable region L11
<400>30
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln SerLeu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>31
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized light chain variable region L12
<400>31
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>32
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized light chain variable region L13
<400>32
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
3540 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>33
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized light chain variable region L15
<400>33
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>34
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized heavy chain variable region H51
<400>34
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>35
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized heavy chain variable region H52
<400>35
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser ThrAla Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>36
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized heavy chain variable region 53
<400>36
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
6570 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>37
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized heavy chain variable region H54
<400>37
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>38
<211>221
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized antibody light chain 38
<400>38
Arg Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
1 5 10 15
Gly Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn
20 25 30
Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly
35 40 45
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
50 55 60
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
65 70 75 80
Thr Ile Ser Ser Leu Gln Ala Glu AspVal Ala Val Tyr Tyr Cys Gln
85 90 95
Asn Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu
100 105 110
Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
115 120 125
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
130 135 140
Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
145 150 155 160
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
165 170 175
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
180 185 190
Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
195 200 205
Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210>39
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized antibody heavy chain 39
<400>39
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>40
<211>220
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized antibody light chain 40
<400>40
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210>41
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized antibody heavy chain 41
<400>41
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>42
<211>220
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized antibody light chain 42
<400>42
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210>43
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized antibody heavy chain 43
<400>43
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>44
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized antibody heavy chain 44
<400>44
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>45
<211>220
<212>PRT
<213>Artificial Sequence
<220>
<223> humanized antibody light chain 45
<400>45
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210>46
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimized light chain variable region L20
<400>46
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu ThrSer
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>47
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimized light chain variable region L21
<400>47
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>48
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimized light chain variable region L22
<400>48
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>49
<211>113
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimized light chain variable region L23
<400>49
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210>50
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimization of heavy chain variable region H60
<400>50
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 5560
Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Thr Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>51
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimization of heavy chain variable region H61
<400>51
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>52
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimization of heavy chain variable region H62
<400>52
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Ile Thr Ala AspLys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>53
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimization of heavy chain variable region H63
<400>53
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>54
<211>118
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimization of heavy chain variable region H64
<400>54
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 7075 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>55
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimized antibody heavy chain
<400>55
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>56
<211>220
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimized antibody light chain
<400>56
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210>57
<211>448
<212>PRT
<213>ArtificialSequence
<220>
<223> deamidation site optimized antibody heavy chain
<400>57
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>58
<211>220
<212>PRT
<213>Artificial Sequence
<220>
<223> deamidation site optimized antibody light chain
<400>58
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210>59
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> Ab43 antibody heavy chain
<400>59
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>60
<211>220
<212>PRT
<213>Artificial Sequence
<220>
<223> Ab24 antibody light chain
<400>60
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210>61
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> Ab24 antibody heavy chain
<400>61
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>62
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> Ab51 antibody heavy chain
<400>62
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
LeuVal Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val LysPhe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His TyrThr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>63
<211>448
<212>PRT
<213>Artificial Sequence
<220>
<223> Ab56 antibody heavy chain
<400>63
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val ThrVal Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe AsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr GlnLys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>64
<211>17
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR3 mutant design 1
<400>64
Lys Ser Ser Gln Ser Leu Leu Thr Ser Gly Asn Asn Lys Asn Tyr Leu
1 5 10 15
Ala
<210>65
<211>17
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR3 mutant design 2
<400>65
Lys Ser Ser Gln Ser Leu Leu Asn Thr Gly Asn Asn Lys Asn Tyr Leu
1 5 10 15
Ala
<210>66
<211>9
<212>PRT
<213>Artificial Sequence
<220>
<223> heavy chain CDR3 mutant design 1
<400>66
Val Tyr Tyr Gly Thr Ser Phe Ala Tyr
1 5
<210>67
<211>10
<212>PRT
<213>Artificial Sequence
<220>
<223> heavy chain CDR3 mutant design 1
<400>67
Ala Arg Val Tyr Tyr Gly Thr Ser Phe Ala
1 5 10
<210>68
<211>9
<212>PRT
<213>Artificial Sequence
<220>
<223> heavy chain CDR3 mutant design 2
<400>68
Val Tyr Tyr Gly Asn Thr Phe Ala Tyr
1 5
<210>69
<211>10
<212>PRT
<213>Artificial Sequence
<220>
<223> heavy chain CDR3 mutant design 2
<400>69
Ala Arg Val Tyr Tyr Gly Asn Thr Phe Ala
1 5 10
<210>70
<211>17
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR1 humanized Var1
<400>70
Lys Ser Ser Gln Ser Val Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu
1 5 10 15
Thr
<210>71
<211>17
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR1 humanized Var2
<400>71
Lys Ser Ser Gln Ser Val Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210>72
<211>17
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR1 humanized Var4
<400>72
Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu
1 5 10 15
Thr
<210>73
<211>17
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR1 humanized Var5
<400>73
Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210>74
<211>17
<212>PRT
<213>Artificial Sequence
<220>
<223> light chain CDR1 humanized Var8
<400>74
Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
Claims (19)
1. A Drug Conjugate (ADC) of an anti-CLDN 18.2 antibody, having the structure of formula I:
Ab-[(L2)n-L1–D]yformula I
Wherein D is small molecular drug with cytotoxicity, L1And L2Separately linking the drug and the antibody; n is 0 or 1; y represents the average number of D coupled to Ab, and 0<y is 10, preferably 2. ltoreq. y.ltoreq.7; more preferably 3. ltoreq. y.ltoreq.6; most preferably 4.4 or 4.8;
the Ab is an antibody capable of specifically binding to human CLDN18.2 comprising a light chain variable region (VL) and/or a heavy chain variable region (VH) in which the anti-CLDN 18.2 antibody correspondingly comprises at least 1 Complementarity Determining Region (CDR) sequence or a mutated sequence thereof selected from the group consisting of:
a VL CDR1 amino acid sequence as shown in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 71 or SEQ ID NO. 74;
a VL CDR2 amino acid sequence set forth in SEQ ID NO. 13;
a VL CDR3 amino acid sequence as set forth in SEQ ID NO. 14;
a VH CDR1 amino acid sequence shown as SEQ ID NO. 15;
the VH CDR2 amino acid sequence shown as SEQ ID NO. 16;
the VH CDR3 amino acid sequence of SEQ ID NO. 17, said mutation maintains or improves binding of said antibody to CLDN 18.2.
2. The drug conjugate of an anti-CLDN 18.2 antibody of claim 1, wherein the small molecule drug is a cytotoxic agent selected from the group consisting of a toxin, a chemotherapeutic agent, an antibiotic, a radioisotope and a nucleolytic enzyme; preferably, the small molecule drug is selected from the group consisting of: monomethyl auristatin (maytansinoid), maytansinoids, camptothecin (camptothecin), calicheamicin, doxorubicin (adriamycin), duocarmycin (duocarmycin), or combinations thereof; more preferably, the monomethyl auristatin is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), and the maytansinoid is N2' -Deacetyl-N2' - (3-mercapto-1-oxopropyl) -maytansine (DM1), N2' -Deacetyl-N2' - (4-mercapto-1-oxopentyl) -maytansine (DM3) and N2' -Deacetyl-N2' - (4-mercapto-4-methyl-1-oxopentyl) -maytansine (DM 4).
3. The drug conjugate of an anti-CLDN 18.2 antibody of claim 1 or 2, wherein L is1Selected from the group consisting of cleavable linkers, non-cleavable linkers, hydrophilic linkers, pre-charged linkers, and dicarboxylic acid-based linkers; preferably, the linker is selected from the group consisting of N-succinimidyl 4- (2-pyridyldithio) valerate (SPP), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), 6-Maleimidocaproyl (MC), Maleimidopropanoyl (MP), valine-citrulline (VC), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and MC-VC-PAB.
4. The drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-3, wherein L is2Is a compound represented by the following formula II:
wherein, X1Selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;
X2selected from alkyl, cycloalkyl and heterocyclyl;
m is 0 to 5; s is a sulfur atom;
preferably, when X1Is a hydrogen atom, X2When m is 1, the compound shown in the formula II is thioacetic acid S- (3-carbonyl propyl) ester.
5. The drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-4, wherein the small molecule drug is DM1 and the linker L is1Is SMCC, n is 0, thereby forming a drug conjugate of the antibody of formula III below:
or, the small molecule drug is MMAF, and the linker L1Is MC-VC-PAB, L2Is S- (3-carbonylpropyl) thioacetate, and n is 1, thereby forming a drug conjugate of the antibody represented by the following formula IV:
6. the drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-5, wherein the anti-CLDN 18.2 antibody comprises a light chain variable region (VL) and/or a heavy chain variable region (VH);
the VL comprises the following amino acid sequence or a mutated sequence thereof:
VL CDR1 shown in SEQ ID NO. 11, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14;
VL CDR1 shown in SEQ ID NO. 12, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14;
VL CDR1 shown in SEQ ID NO:71, VL CDR2 shown in SEQ ID NO:13 and VL CDR3 shown in SEQ ID NO: 14; or
VL CDR1 shown in SEQ ID NO. 74, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14;
the VH comprises the following amino acid sequence or a mutated sequence thereof:
VH CDR1 shown in SEQ ID NO. 15, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17.
7. The drug conjugate of an anti-CLDN 18.2 antibody of claim 6, wherein the anti-CLDN 18.2 antibody comprises a VL and a VH:
the VL comprises an amino acid sequence or a mutant sequence of VL CDR1 shown in SEQ ID NO. 11, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14; the VH comprises the amino acid sequence of VH CDR1 shown in SEQ ID NO. 15, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17 or mutant sequences thereof;
the VL comprises an amino acid sequence or a mutant sequence of VL CDR1 shown in SEQ ID NO. 12, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14; the VH comprises the amino acid sequence of VH CDR1 shown in SEQ ID NO. 15, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17 or mutant sequences thereof;
the VL comprises an amino acid sequence or a mutant sequence of VL CDR1 shown in SEQ ID NO. 71, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14; the VH comprises the amino acid sequence of VH CDR1 shown in SEQ ID NO. 15, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17 or mutant sequences thereof;
the VL comprises an amino acid sequence or a mutant sequence of VL CDR1 shown in SEQ ID NO. 74, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14; the VH comprises the amino acid sequence of VH CDR1 shown in SEQ ID NO. 15, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17 or mutant sequences thereof;
the VL comprises an amino acid sequence or a mutant sequence of VL CDR1 shown in SEQ ID NO. 11, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14; the VH comprises the amino acid sequence of VH CDR1 shown in SEQ ID NO. 18, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17 or mutant sequences thereof;
the VL comprises an amino acid sequence or a mutant sequence of VL CDR1 shown in SEQ ID NO. 12, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14; the VH comprises the amino acid sequence of VH CDR1 shown in SEQ ID NO. 18, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17 or mutant sequences thereof;
the VL comprises an amino acid sequence or a mutant sequence of VL CDR1 shown in SEQ ID NO. 71, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14; the VH comprises the amino acid sequence of VH CDR1 shown in SEQ ID NO. 18, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17 or mutant sequences thereof; or
The VL comprises an amino acid sequence or a mutant sequence of VL CDR1 shown in SEQ ID NO. 74, VL CDR2 shown in SEQ ID NO. 13 and VL CDR3 shown in SEQ ID NO. 14; the VH comprises the amino acid sequence of VH CDR1 shown in SEQ ID NO. 18, VH CDR2 shown in SEQ ID NO. 16 and VH CDR3 shown in SEQ ID NO. 17 or mutant sequences thereof.
8. The drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-7, wherein the anti-CLDN 18.2 antibody is a murine antibody;
preferably, the amino acid sequence of the variable region of the light chain of the murine antibody is the amino acid sequence shown as SEQ ID NO. 7 or a mutant sequence thereof; and/or the heavy chain variable region amino acid sequence of the murine antibody is the amino acid sequence shown as SEQ ID NO. 8 or a mutant sequence thereof.
9. The drug conjugate of an anti-CLDN 18.2 antibody of claim 8, wherein the anti-CLDN 18.2 antibody comprises a variable region of a murine antibody and a constant region of a murine or human antibody; the murine antibody constant regions comprise the heavy chain constant region of murine IgG1, IgG2a, IgG2b3, or IgG3 and light chain constant regions of the kappa or lambda type; the human antibody constant regions comprise a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, and a light chain constant region of kappa or lambda type;
preferably, the anti-CLDN 18.2 antibody is a chimeric antibody synthesized from the variable region of a murine antibody and the constant region of a human antibody;
more preferably, the light chain amino acid sequence of the chimeric antibody is the amino acid sequence shown in SEQ ID NO. 9 or a mutant sequence thereof; and/or the heavy chain amino acid sequence of the chimeric antibody is the amino acid sequence shown in SEQ ID NO. 10 or a mutant sequence thereof.
10. The drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-7, wherein the anti-CLDN 18.2 antibody is a humanized antibody;
preferably, the light chain variable region sequence of the humanized antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs 29-33 or a mutated sequence thereof; and/or the heavy chain variable region sequence of the humanized antibody comprises an amino acid sequence shown in any one of SEQ ID NO 34-37 or a mutant sequence thereof.
11. The drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-10, wherein the CDR mutation sequence of the anti-CLDN 18.2 antibody is a sequence having a mutation at a deamination sensitive site in a CDR region;
preferably, the CDR regions are deamination sensitive sites of the light chain CDR1 at L30A and/or L30B; and/or, heavy chain CDR3 at positions H99 and/or H100;
most preferably, the light chain variable region sequence of the sequence in which the human CDR region is subject to a deamination sensitive site mutation comprises the amino acid sequence shown in any one of SEQ ID Nos. 46-49 or a mutated sequence thereof; and/or the heavy chain variable region sequence of the human CDR region with mutation of deamination sensitive site comprises the amino acid sequence shown in any one of SEQ ID NO 50-54 or a mutant sequence thereof.
12. The drug conjugate of an anti-CLDN 18.2 antibody of claim 10 or 11, wherein the light chain of the antibody comprises a constant region selected from human kappa or lambda type light chains or variants thereof; and/or the heavy chain of the antibody comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3, and IgG4, or a variant thereof;
preferably, the heavy chain constant region or variant thereof comprises the mutations at position 243, or at positions 239, 330 and 332 of the human IgG1Fc region;
more preferably, the heavy chain constant region or variant thereof comprises a variant of the human IgG1Fc region at positions 356-358 of EEM or DEL.
13. The drug conjugate of the anti-CLDN 18.2 antibody of any one of claims 10-12, wherein the light chain of the antibody comprises the amino acid sequence shown as SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 45, SEQ ID No. 56, SEQ ID No. 58, or SEQ ID No. 60, or a light chain sequence having at least 85% sequence homology thereto; and/or the presence of a gas in the gas,
the heavy chain of the antibody comprises an amino acid sequence shown by SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 62 or SEQ ID NO 63 or a heavy chain sequence with at least 85 percent of sequence homology with the amino acid sequence;
said at least 85% sequence homology is preferably at least 90% sequence homology; more preferably at least 95% sequence homology; most preferably at least 99% sequence homology.
14. The drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-13, wherein the anti-CLDN 18.2 antibody comprises the amino acid sequences of the following light and heavy chains:
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 39, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 38;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 39, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 40;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 41, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 38;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 41, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 40;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 39, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 42;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 43, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 42;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 44, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 42;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 43, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 45;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 44, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 45;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 39, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 45;
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 55, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 38; or the like, or, alternatively,
the heavy chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 55, and the light chain amino acid sequence of the anti-CLDN 18.2 antibody is shown as SEQ ID NO. 42.
15. The drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-14, wherein the anti-CLDN 18.2 antibody comprises a half-antibody or an antigen-binding fragment of a half-antibody, preferably, a Fab, Fab ', F (ab')2, Fv or single chain Fv fragment (scFv).
16. The drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-13, which is a drug conjugate of an antibody represented by the following formula V:
or, a drug conjugate of an antibody of formula VI below:
or, a drug conjugate of an antibody represented by formula VII below:
or, a drug conjugate of an antibody represented by formula VIII below:
wherein Ab10 comprises a light chain as shown in SEQ ID NO. 38 and a heavy chain as shown in SEQ ID NO. 39; the Ab6 contains a light chain as shown in SEQ ID NO. 42 and a heavy chain as shown in SEQ ID NO. 39.
17. A pharmaceutical composition comprising a drug conjugate of the anti-CLDN 18.2 antibody of any one of claims 1-16 and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further comprises an immune checkpoint antibody.
18. A method for preparing a drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1-16,
when n is 1, the preparation method comprises the following steps:
(1) preparation of intermediate 1: taking the antibody and the linker L2Mixing the mixture in the solution, reacting and purifying to obtain the solution containing the intermediate 1, wherein the intermediate 1 is a compound of formula IIntermediate 1 is represented by formula IX below:
wherein, X1Selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;
X2selected from alkyl, cycloalkyl and heterocyclyl;
m is 0 to 5; s is a sulfur atom;
preferably, X1Is a hydrogen atom, X2Is alkyl, m is 1, i.e. L2Is thioacetic acid S- (3-carbonylpropyl) ester;
(2) preparation of intermediate 2: taking out the joint L1And the drug is prepared into an intermediate 2: l is1-D;
(3) Mixing the solution containing the intermediate 1 obtained in the step (1) with the solution containing the intermediate 2 obtained in the step (2), reacting and purifying to obtain a solution of a drug conjugate containing the antibody;
when n is 0, the preparation method comprises the following steps:
(1) preparation of intermediate 3: taking the antibody and the linker L1Mixing the mixture in the solution, reacting and purifying to obtain a solution containing an intermediate 3;
(2) mixing the solution containing the intermediate 3 obtained in the step (1) with the solution containing the drug, reacting and purifying to obtain a solution of a drug conjugate containing the antibody;
preferably, in the step (1) and/or (2), the reaction temperature is 25 ℃; the reaction time is 2-4 hours; and/or, the purification is purification by gel filtration, more preferably purification by desalting on a Sephadex G25 gel column.
19. Use of the drug conjugate of an anti-CLDN 18.2 antibody of any one of claims 1 to 16 and the pharmaceutical composition of claim 17 for the preparation of a medicament for the prevention or treatment of tumors; preferably, the tumor is lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, leukemia, or the like, or a metastatic lesion thereof.
Priority Applications (6)
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CN201811295845.2A CN111110862A (en) | 2018-11-01 | 2018-11-01 | Drug conjugate of anti-CLDN 18.2 antibody, preparation method and application thereof |
US17/252,259 US11912763B2 (en) | 2018-06-17 | 2019-06-06 | Antibody targeting CLDN18.2, bispecific antibody, ADC, and CAR, and applications thereof |
EP19823355.3A EP3808376A4 (en) | 2018-06-17 | 2019-06-06 | Antibody targeting cldn18.2, bispecific antibody, adc, and car, and applications thereof |
CN201980019477.5A CN111867630B (en) | 2018-06-17 | 2019-06-06 | Antibodies targeting CLDN18.2, bispecific antibodies, ADCs and CARs and uses thereof |
PCT/CN2019/090255 WO2019242505A1 (en) | 2018-06-17 | 2019-06-06 | Antibody targeting cldn18.2, bispecific antibody, adc, and car, and applications thereof |
JP2020571669A JP7468903B2 (en) | 2018-06-17 | 2019-06-06 | Antibodies, bispecific antibodies, ADCs and CARs targeting CLDN18.2 and uses thereof |
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