Anti-CLDN18.2 antibody conjugate

Abstract

The present disclosure provides herein conjugates of anti-CLDN18.2 antibodies or antigen-binding fragments thereof with radionuclides, pharmaceutical compositions containing same, and uses thereof in imaging, patient screening, treatment process monitoring and efficacy evaluation.

Description

[Technical field]

[0001] The present invention relates to nuclear medicine, in particular to the preparation and application of radiolabeled CLDN18.2 antibody conjugates. [Background technology]

[0002]

[0002] In the world, the incidence and deaths of gastric cancer ranked fifth and third, respectively. The incidence of gastric cancer ranked second in China. Currently, trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2), is the only drug with a survival benefit in the first-line treatment of advanced gastric cancer. In recent years, good news about new drugs targeting HER2 (antibody-drug conjugates, bispecific antibodies, small molecule inhibitors, etc.) has been frequently reported, but the proportion of HER2-positive gastric cancer patients is low (only 10-12%), most of them develop drug resistance within one year, and the troublesome situation of a lack of precise and effective targeted drugs after drug resistance still cannot be wiped away. Therefore, exploring new targets for gastric cancer treatment is the key to break through the bottleneck of advanced gastric cancer treatment.

[0003]

[0003] The 2016 FAST study validated Claudin18.2 (claudin18 splice variant 2, CLDN18.2) as a "nova" target in gastric cancer through a series of experiments with IMAB362, a monoclonal antibody targeting CLDN18.2. However, to date, no reports have been published on anti-CLDN18.2 nuclide probes. Summary of the Invention [Problem to be solved by the invention]

[0004]

[0004] It is significant to develop a molecular probe with high specificity for the diagnosis of CLDN18.2 by using the comprehensive, non-invasive, real-time and dynamic characteristics of modern molecular imaging technology. Therefore, there is a great need for novel anti-CLDN18.2 nuclide probes and therapies.

[0005]

[0005] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or more than one antibody. [Means for solving the problem]

[0006]

[0006] In one aspect, the present disclosure provides an anti-CLDN18.2 antibody conjugate comprising an anti-CLDN18.2 antibody or an antigen-binding fragment thereof conjugated to a radionuclide, including a therapeutic radionuclide or a diagnostic radionuclide.

[0007] In some embodiments, the therapeutic radionuclide is 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra,225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb.

[0008] In some embodiments, the diagnostic radionuclide is 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 l, 124 l, 125 l, 131 l, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154’’1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re,188 Re, 189 Re, 194 lr, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac.

[0009]

[0009] In some embodiments, the diagnostic radionuclide is detectable by positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0010] In some embodiments, the radionuclide is 64 Cu, 67 Cu, 89 Zr, 124 I, 86 Y, 90 Y, 111 In, 123 / 131 I, 177 Lu, 11 C. 14 C. 41 Ca, 67 Ga, 68 Ga, 13 N, 15 O. 44 Sc, 18 F, 99m Tc, and 90m Tc.

[0011] In some embodiments, the radionuclide is 124 I or 123 I or 131 I.

[0012] In some embodiments, the radionuclide is 64 Cu, 67 Cu, or 89 Zr.

[0013] In some embodiments,64 Cu, 67 Cu, or 89 Zr is labeled to the antibody or its antigen-binding fragment via a chelating agent.

[0014] In some embodiments, the chelating agent comprises three or more atoms for chelation, each atom being selected from the group consisting of nitrogen, sulfur, oxygen, and phosphorus.

[0015] In some embodiments, the chelating agent comprises DFO (delferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (NR-diethylenetriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-acetic acid), 1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid (abbreviated herein as TRITA); 1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (abbreviated herein as TETA); and 1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid (abbreviated herein as HETA), ethylenediaminetetraacetic acid (abbreviated herein as EDTA), or diethylenetriaminepentaacetic acid (DTPA).

[0016] In some embodiments, the radionuclide is 64 Cu or 67 Cu, the chelating agent comprises TETA, NOTA, NODA, or NODGA, or the radionuclide is 89 Zr and the chelating agent comprises DFO.

[0017] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises the sequences of heavy chain HCDR1, HCDR2, and HCDR3, and / or light chain LCDR1, LCDR2, and LCDR3, wherein: the HCDR1 sequence comprises GYNMN (SEQ ID NO:1), or TYFIGVG (SEQ ID NO:13), or a homologous sequence thereof with at least 80% sequence identity; the HCDR2 sequence comprises X1IDPYYX2X3TX4YNQKFX5G (SEQ ID NO: 32), or HIWWNDNKYYNTALKS (SEQ ID NO: 15), or a homologous sequence thereof with at least 80% sequence identity; the HCDR3 sequence comprises X6X7X8GNAFDY (SEQ ID NO:33), or MGSGAWFTY (SEQ ID NO:17), or a homologous sequence of at least 80% sequence identity thereof; The LCDR1 sequence is KSSQX9LX 10 NX 11 GNX 12 KNYLT (SEQ ID NO: 34), or a homologous sequence thereof with at least 80% sequence identity; LCDR2 array is WASTRX 13 S (SEQ ID NO:35), or a homologous sequence having at least 80% sequence identity thereto; LCDR3 array is QNDYX 14 X 15 PX 16 T (SEQ ID NO: 36), or a homologous sequence having at least 80% sequence identity thereto; where X1 is N or Y or H, X2 is G or V, X3 is A or G or T, X4 is R or T or S, X5 is K or R, X6 is S or M, X7 is Y or F, X8 is Y or H, X9 is S or N, and X 10 is L or F, and X 11 is S or N, and X 12 is Q or L, and X 13 is E or K, and X 14 is S or Y, and X 15 is F or Y and X 16 is F or L.

[0018] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof is: a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:3, and an HCDR3 comprising the sequence of SEQ ID NO:5; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:7, and an HCDR3 comprising the sequence of SEQ ID NO:5; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:8; a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:9, and an HCDR3 comprising the sequence of SEQ ID NO:11; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:10, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:13, an HCDR2 comprising the sequence of SEQ ID NO:15, and an HCDR3 comprising the sequence of SEQ ID NO:17; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:12; a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:19, and an HCDR3 comprising the sequence of SEQ ID NO:21; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:16, and an LCDR3 comprising the sequence of SEQ ID NO:18; or a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:22, and an HCDR3 comprising the sequence of SEQ ID NO:5; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:20, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6. Includes.

[0019] In some embodiments, the heavy chain variable region further comprises one or more of heavy chains HFR1, HFR2, HFR3, and HFR4, and / or the light chain variable region further comprises one or more of light chains LFR1, LFR2, LFR3, and LFR4, wherein: HFR1 is QVQLVQSGAEVKKPGASVKVSCKASGYX 17 FT (SEQ ID NO: 54) or a homologous sequence thereof with at least 80% sequence identity; HFR2 is WVX 18 QAPGQGLEWX 19 G (SEQ ID NO:55) or a homologous sequence thereof with at least 80% sequence identity; The HFR3 sequence is RVTX 20 TIDKSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO:56), or a homologous sequence thereof with at least 80% sequence identity; HFR4 comprises WGQGTTVTVSS (SEQ ID NO:57) or a homologous sequence thereof of at least 80% sequence identity; LFR1 is DIVMTQSPDSLAVSLGERATX 21 NC (SEQ ID NO:58) or a homologous sequence thereof with at least 80% sequence identity; LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO:59) or a homologous sequence thereof of at least 80% sequence identity; LFR3 is GVPDRFX 22 GSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 60) or a homologous sequence of at least 80% sequence identity thereto; LFR4 comprises FGGGTKVEIK (SEQ ID NO: 61) or a homologous sequence thereof with at least 80% sequence identity; Where X 17 is T or S, and X 18 is R or K, and X 19 is M or I, and X 20 is M or L, and X 21 is I or M, and X 22 is S or T.

[0020] In some embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 62 and 63; HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 64 and 65; HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 66 and 67; HFR4 comprises the sequence of SEQ ID NO:57, LFR1 comprises a sequence from the group consisting of SEQ ID NOs: 68 and 69; LFR2 comprises the sequence of SEQ ID NO:59, LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 70 and 71; LFR4 comprises the sequence of SEQ ID NO:61.

[0021]

[0021] In some embodiments, the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47, and homologous sequences thereof having at least 80% sequence identity while still retaining specific binding affinity to CLDN18.2.

[0022]

[0022] In some embodiments, the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, and homologous sequences thereof having at least 80% sequence identity while still retaining specific binding affinity to CLDN18.2.

[0023] In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO:23 and the light chain variable region comprises the sequence of SEQ ID NO:24; the heavy chain variable region comprises the sequence of SEQ ID NO:25 and the light chain variable region comprises the sequence of SEQ ID NO:26; the heavy chain variable region comprises the sequence of SEQ ID NO:27 and the light chain variable region comprises the sequence of SEQ ID NO:28; the heavy chain variable region comprises the sequence of SEQ ID NO:29 and the light chain variable region comprises the sequence of SEQ ID NO:26 or 28; the heavy chain variable region comprises the sequence of SEQ ID NO:37 and the light chain variable region comprises the sequence of SEQ ID NO:38; the heavy chain variable region comprises the sequence of SEQ ID NO:39 and the light chain variable region comprises the sequence of SEQ ID NO:40; the heavy chain variable region comprises the sequence of SEQ ID NO:41 and the light chain variable region comprises the sequence of SEQ ID NO:42; the heavy chain variable region comprises the sequence of SEQ ID NO:43 and the light chain variable region comprises the sequence of SEQ ID NO:44; the heavy chain variable region comprises the sequence of SEQ ID NO:45 and the light chain variable region comprises the sequence of SEQ ID NO:46; or The heavy chain variable region comprises the sequence of SEQ ID NO:47, and the light chain variable region comprises the sequence of SEQ ID NO:48.

[0024]

[0024] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof further comprises a substitution or modification of one or more amino acid residues that still retains specific binding affinity to human CLDN18.2.

[0025]

[0025] In some embodiments, at least one of the substitutions or modifications is in one or more CDR sequences and / or in one or more non-CDR regions of the VH or VL sequences.

[0026]

[0026] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region.

[0027]

[0027] In some embodiments, the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0028]

[0028] In some embodiments, the constant region of human IgG1 comprises SEQ ID NO: 49, or a homologous sequence having at least 80% sequence identity thereto.

[0029]

[0029] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof is humanized.

[0030]

[0030] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single domain antibody, nanobody, domain antibody, and bivalent domain antibody.

[0031]

[0031] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody conjugate provided herein and one or more pharma ceutically acceptable carriers.

[0032]

[0032] In some embodiments, the radiochemical purity is at least 95% (eg, 97%, 98%, 99%).

[0033]

[0033] In another aspect, the present disclosure provides a method of obtaining an image of a region of interest in a subject, the method comprising: a) administering to a subject an effective amount of an antibody conjugate provided herein and / or a pharmaceutical composition provided herein; b) subjecting the area of ​​interest of the subject to positron emission tomography (PET) or SPECT; c) identifying a detectable signal from the radionuclide in the subject; d) imaging the detectable signal, thereby obtaining an image of the site of interest in the subject; The present invention provides a method comprising:

[0034]

[0034] In some embodiments, the site of interest is a site that expresses or is suspected of expressing claudin18.2.

[0035]

[0035] In some embodiments, the site of interest contains or is suspected of containing a tumor.

[0036] In another aspect, the present disclosure provides a method for detecting or visualizing claudin18.2 expression in a subject in a non-invasive manner, comprising: a) administering to a subject an effective amount of an antibody conjugate provided herein and / or a pharmaceutical composition provided herein; b) subjecting the subject to positron emission tomography (PET) or SPECT; c) identifying a detectable signal from the radionuclide at a site of interest in the subject; d) determining or visualizing claudin18.2 expression in the site of interest of the subject based on the identified detectable signal; The present invention provides a method comprising:

[0037]

[0037] In some embodiments, the methods further comprise administering a therapeutically effective amount of an anti-claudin18.2 therapy to the subject identified as having claudin18.2 expression at the site of interest.

[0038]

[0038] In some embodiments, the method further comprises determining a distribution of claudin18.2 expression in the site of interest of the subject based on the identified detectable signal.

[0039]

[0039] In some embodiments, the method further comprises determining the heterogeneity of claudin18.2 expression in the subject's site of interest.

[0040]

[0040] In some embodiments, the site of interest is a tumor.

[0041] In another aspect, the present disclosure provides a method for monitoring, in a non-invasive manner, therapeutic efficacy, responsiveness to treatment, or the occurrence of resistance or recurrence, or metastasis, in a subject who has been treated for a therapeutic period, comprising: a) administering to a subject an effective amount of an antibody conjugate provided herein and / or a pharmaceutical composition provided herein; b) subjecting the subject to positron emission tomography (PET) or SPECT; c) identifying a detectable signal from the radionuclide at a site of interest in the subject; d) determining post-treatment claudin18.2 expression at the site of interest in the subject based on the identified detectable signal; e) comparing the level or distribution of claudin18.2 expression after treatment to a baseline level or distribution of claudin18.2 expression obtained from the subject prior to a treatment period, respectively, to determine a post-treatment change in the level or distribution of claudin18.2 expression in the subject; f) determining therapeutic efficacy, responsiveness to treatment, or occurrence of resistance or recurrence, or metastasis based on the changes determined in step (e); The present invention provides a method comprising:

[0042]

[0042] In some embodiments, baseline claudin18.2 expression levels or distribution are determined using similar methods.

[0043] In some embodiments, the baseline claudin18.2 expression level or distribution is determined before the treatment period (i.e., before treatment) by administering an effective amount of the antibody conjugate provided herein and / or pharmaceutical composition provided herein to the subject, subjecting the subject to positron emission tomography (PET) or SPECT, identifying a detectable signal from a radionuclide at the site of interest of the subject, and determining the baseline claudin18.2 expression at the site of interest of the subject based on the identified detectable signal. In some embodiments, the site of interest for determining the baseline claudin18.2 expression is the same as or at least equivalent to the site of interest for determining the claudin18.2 expression after treatment.

[0044]

[0044] In some embodiments, elevated levels or spread of claudin18.2 expression indicates reduced therapeutic efficacy, reduced responsiveness to treatment, or the presence of resistance or relapse.

[0045]

[0045] In some embodiments, the subject has cancer.

[0046]

[0046] In another aspect, the present disclosure provides a method for treating a CLDN18.2-related disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an anti-CLDN18.2 antibody conjugate provided herein and / or a pharmaceutical composition provided herein.

[0047]

[0047] In some embodiments, the disease or condition is cancer, optionally a CLDN18.2-expressing cancer.

[0048]

[0048] In some embodiments, the subject has been identified as having a CLDN18.2-expressing cancer.

[0049]

[0049] In another aspect, the present disclosure provides a kit comprising an anti-CLDN18.2 antibody conjugate provided herein, or a composition provided herein.

[0050]

[0050] In another aspect, the present disclosure provides a kit comprising a first anti-CLDN18.2 antibody conjugate provided herein and a second anti-CLDN18.2 antibody conjugate provided herein, wherein the first anti-CLDN18.2 antibody conjugate comprises a diagnostic radionuclide and the second anti-CLDN18.2 antibody conjugate comprises a therapeutic radionuclide.

[0051]

[0051] In some embodiments, the kits are for use in the methods provided herein.

[0052]

[0052] In another aspect, the present disclosure provides the use of an anti-CLDN18.2 antibody conjugate provided herein or a composition provided herein, wherein the anti-CLDN18.2 antibody conjugate comprises a diagnostic radionuclide, in the manufacture of a medicament for use in a method provided herein for obtaining an image of a site of interest in a subject, or for use in a method provided herein for detecting or visualizing claudin18.2 expression in a subject in a non-invasive manner, or for use in a method provided herein for monitoring therapeutic efficacy, responsiveness to treatment, or the occurrence of resistance or recurrence, or metastasis in a subject in a non-invasive manner, wherein the anti-CLDN18.2 antibody conjugate comprises a diagnostic radionuclide.

[0053]

[0053] In another aspect, the present disclosure provides the use of an anti-CLDN18.2 antibody conjugate provided herein or a composition provided herein in the manufacture of a medicament for use in a method for treating a CLDN18.2-associated disease or condition in a subject in need of such treatment, wherein the anti-CLDN18.2 antibody conjugate comprises a therapeutic radionuclide.

[0054] In another aspect, the present disclosure provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising: 124 I, 123 I or 131 A method for preparing an anti-CLDN18.2 antibody conjugate provided herein is provided, comprising reacting an iodide compound labeled with I in the presence of an enzymatic or chemical oxidizing agent.

[0055] In some embodiments, the chemical oxidizing agent is N-bromosuccinimide, iodogen, or chloramine-T.

[0056] In another aspect, the present disclosure provides a method for the preparation of a CLDN18.2 antibody, or antigen-binding fragment thereof, comprising the steps of: conjugating an anti-CLDN18.2 antibody, or antigen-binding fragment thereof, with a chelator to obtain a chelator-antibody conjugate; 64 Cu or 89 and reacting the anti-CLDN18.2 antibody conjugate provided herein with Zr.

[0057] In some embodiments, the chelating agent is DFO (delferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (NR-diethylenetriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-acetic acid), TRITA (1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid); TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid); and HETA (1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid), NETA ({4-[2-(bis-carboxymethylamino)-ethyl]]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), TACN-TM (N,N',N",-tris(2-mercaptoethyl)1,4,7-triazacyclononane), TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]), CP256, PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid), porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and derivatives thereof. [Brief description of the drawings]

[0058] [Figure 1]

[0058] Figure 1 shows the radiolabeling rate and radiochemical purity of the 124I-18B10 probe: A) radiochemical purity after purification; B) radiochemical purity after purification; C) radiochemical purity after 1 hour in PBS; D) radiochemical purity after 48 hours in PBS. [Diagram 2]

[0059] FIG. 2 shows the uptake of 124I-18B10 and its control in MKN45-CLND18.2 high / MKN45 cells. [Diagram 3]

[0060] FIG. 3 shows the biodistribution of 124I-18B10 in normal Kunming mice. [Figure 4]

[0061] FIG. 4 shows a comparison of Micro-PET imaging of 124I-18B10 and its control group as well as 18F-FDG in CLDN18.2 positive / negative PDX models of Example 5 of the present invention. [Figure 5A]

[0062] 5A-B are bar graphs showing the binding signals of chimeric antibodies to mutant hCLDN18.2 variants using epitope mapping. When E56 was mutated to Q, the binding of 18B10-C was completely lost. This change was also true for other chimeric antibodies except for IMAB362 and 59A9-C. Other amino acids such as A42, N45 also contributed to some extent to the binding of IMAB362 and other antibodies, but not for 18B10-C. [Figure 5B]

[0062] Figures 5A-B are bar graphs showing binding signals of chimeric antibodies to mutated hCLDN18.2 variants using epitope mapping. [Figure 6]

[0063] FIG. 6 shows the biodistribution of 124I-18B10 in PDX mice. [Figure 7]

[0064] FIG. 7 shows the biodistribution of 124I-18B10 in patients, derived from images of the first five patients. [Figure 8]

[0065] FIG. 8 shows the accumulation of 124I-18B10 in the ovarian lesions of patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059]

[0066] The following description of the present disclosure is intended to merely illustrate various embodiments of the present disclosure. As such, the specific modifications discussed should not be interpreted as limitations on the scope of the present disclosure. It is clear to those skilled in the art that various equivalents, variations and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included in this specification. All references cited in this specification, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0060] definition

[0067] As used herein, the terms "a," "an," "the," and similar terms as used in the context of the present invention (particularly in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0061]

[0068] The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A naturally occurring intact antibody contains two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain contains a variable region (V H ) and the first, second, and third constant regions (C H1 , C H2 , C H3 Mammalian light chains are classified as λ or κ, and each light chain is composed of a variable region (V L) and constant regions. Antibodies have the shape of a "Y" with the stem of the Y being composed of the second and third constant regions of two heavy chains bound together via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3). The boundaries of the CDRs of the antibodies and antigen binding domains disclosed herein may be determined according to the Kabat, IMGT, AbM, Chothia, or Al-Lazikani convention (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec. 5, 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987); N.R. Whitelegg et al., Protein. Engineering, vol. 13(12), pp. 819-824 (2000); Chothia, C. et al., Nature. Dec. 21-28; 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, pp. 481-514, (2015)). The three CDRs are interposed between adjacent stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops.The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the main antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain). In certain embodiments, the antibodies provided herein include any antigen-binding fragment thereof.

[0062]

[0069] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a fragment of an antibody that contains one or more CDRs, or any other antibody portion that binds to an antigen but does not contain an intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen that the parent antibody binds. In certain embodiments, antigen-binding fragments can include one or more CDRs from a particular human antibody.

[0063]

[0070] "Fab" with respect to an antibody refers to a monovalent antigen-binding fragment of an antibody that is composed of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain. Fabs can be obtained by papain digestion of an antibody at residues in the hinge region proximal to the N-terminus of the disulfide bond between the heavy chains.

[0064]

[0071] "Fab'" refers to a Fab fragment containing a portion of the hinge region, which may be obtained by pepsin digestion of an antibody at the residues proximal to the C-terminus of the inter-heavy chain disulfide bond in the hinge region, and which therefore differs from Fab in a small number of residues in the hinge region, including one or more cysteines.

[0065]

[0072] "F(ab')2" refers to a dimer of Fab' containing two light chains and part of two heavy chains.

[0066]

[0073] "Fc" with respect to an antibody refers to the portion of the antibody that consists of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonds. The Fc region of IgG and IgM contains three heavy chain constant regions (the second, third and fourth heavy chain constant regions in each chain). It can be obtained by papain digestion of the antibody. The Fc portion of an antibody is responsible for various effector functions such as ADCC, ADCP and CDC, but does not function in antigen binding.

[0067]

[0074] "Fv" with respect to an antibody refers to the smallest fragment of an antibody that retains a complete antigen-binding site. An Fv fragment is composed of the variable region of a single light chain bound to the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment, in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0068]

[0075] "Single chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region and a heavy chain variable region connected to each other directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)). "scFv dimer" refers to a single chain containing two heavy chain variable regions and two light chain variable regions with a linker. In certain embodiments, an "scFv dimer" is a bivalent diabody or a bivalent ScFv (BsFv), which is a V H A part of VL and to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L The V dimerized with the moiety H -V L (linked by a peptide linker). In another embodiment, an "scFv dimer" is a bispecific diabody, which comprises H1 and V L1 V H2 and V L2 V is arranged so that they cooperate and each cooperated pair has a different antigen specificity. L1 -V H2 (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0069]

[0076] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody composed of an scFv attached to the Fc region of an antibody.

[0070]

[0077] "Camelized single domain antibodies", "heavy chain antibodies", "nanobodies" or "HCAbs" are antibodies that consist of two V HIt refers to antibodies that contain heavy chain domains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec. 10; 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun. 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally obtained from the Camelidae family (camels, dromedaries and llamas). Although lacking light chains, camelized antibodies have a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3;363(6428):446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. April;54(1):39-47 (2002); Nguyen VK. et al. Immunology. May;109(1):93-101 (2003)). The variable domains of heavy-chain antibodies (VHH domains) represent the smallest known antigen-binding units generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8. Epub 2007 June 15 (2007)). "Diabodies" include small antibody fragments with two antigen-binding sites, where the fragments are arranged in a single polypeptide chain. L V connected to the domain H Domain (V H -V L or V L -V H) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90(14):6444-8 (1993); EP404097; WO93 / 11161). The two domains on the same chain cannot pair because the linker is too short, so they are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The two antigen-binding sites can target the same or different antigens (or epitopes).

[0071]

[0078] A "domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or only the variable region of a light chain. In certain embodiments, two or more V H The domains are covalently linked with a peptide linker to form a bivalent or multivalent domain antibody. H The domains can target the same or different antigens.

[0072]

[0079] In certain embodiments, a "(dsFv)2" is a Fv comprising three peptide chains: a Fv comprising two V L Two V's joined together H Includes parts.

[0073]

[0080] In certain embodiments, a "bispecific ds diabody" is H1 and V L1 Through a disulfide bridge between V L1 -V H2 V bound to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker) Includes.

[0074]

[0081] In certain embodiments, a "bispecific dsFv" or "dsFv-dsFv'" comprises three peptide chains: H1 -V H2The two heavy chains are linked by a peptide linker (e.g., a long flexible linker) and are connected via disulfide bridges to each other by V L1 Part and V L2 Partially paired, V H1 -V H2 Each disulfide-paired heavy and light chain has a different antigen specificity.

[0075]

[0082] The term "humanized" as used herein means that the antibody or antigen-binding fragment comprises CDRs derived from a non-human animal, FR regions derived from a human, and, if applicable, a constant region derived from a human. In certain embodiments, amino acid residues in the variable region framework of a humanized CLDN18.2 antibody are replaced for sequence optimization. In certain embodiments, the variable region framework sequences of the humanized CLDN18.2 antibody chains are at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the corresponding human variable region framework sequences.

[0076]

[0083] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of the heavy and / or light chain derived from one species and the remaining portion of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0077]

[0084] The term "germline sequence" refers to a nucleic acid sequence encoding a variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison with all other known variable region amino acid sequences encoded by germline immunoglobulin variable region sequences. A germline sequence can also refer to a variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison with all other evaluated variable region amino acid sequences. A germline sequence may be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that include variable regions. Sequence identity can be determined by aligning the two sequences using the methods described herein, for example, BLAST, ALIGN, or another alignment algorithm known in the art. A germline nucleic acid or amino acid sequence can have at least about 90%, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference variable region nucleic acid or amino acid sequence. Germline sequences can be determined, for example, through the publicly available international ImMunoGeneTics database (IMGT) and V-base.

[0078]

[0085] As used herein, "anti-CLDN18.2 antibody" or "antibody to CLDN18.2" refers to an antibody that can specifically bind to CLDN18.2 (e.g., human or non-human CLDN18.2) with sufficient affinity to provide, for example, diagnostic and / or therapeutic uses.

[0079]

[0086] The term "affinity" as used herein refers to the strength of the non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen.

[0080]

[0087] As used herein, the terms "specific binding" or "specifically bind" refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein have a specific binding affinity of ≦10 -6 M (e.g., ≦5×10 -7 M, ≤2×10 -7 M, ≦10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≦10 -8 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M,≦2×10 -9 M, or ≤ 10 -9 Binding affinity (K D ) specifically binds to human and / or non-human CLDN18.2. D is the ratio of the dissociation rate to the association rate (k off / k on ), which may be determined by using any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). In certain embodiments, K D Values ​​may be suitably determined by using flow cytometry techniques. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that may be used to determine specific immunoreactivity). Usually, a specific or selective binding reaction will produce a signal that is at least 2-fold over background, more usually at least 10-100-fold over background.

[0081]

[0088] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to obtain maximum correspondence. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be performed using publicly available tools such as, for example, BLASTN, BLASTp (available at the US National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters, such as by selecting an appropriate algorithm, as necessary for alignment. In certain embodiments, non-identical residue positions may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art.See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference.

[0082]

[0089] As used herein, "homolog sequence" and "homologous sequence" are used interchangeably and refer to a polynucleotide sequence (or its complementary strand) or amino acid sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence, optionally when aligned.

[0083]

[0090] An "isolated" material is one that has been altered by the hand of man from its natural state. An "isolated" composition or material, when present in nature, has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated", but is "isolated" when the same polynucleotide or polypeptide is sufficiently separated from the coexisting materials of its natural state so that it exists in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic (e.g., ion exchange chromatography or reverse phase HPLC) methods.

[0084]

[0091] As used herein, the ability to "block binding" or "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human CLDN18.2 and an anti-CLDN18.2 antibody) to any detectable extent. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0085]

[0092] As used herein, the term "antibody conjugate" refers to the linkage of an antibody or antigen-binding fragment thereof to another agent, such as a radionuclide.

[0086]

[0093] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0087]

[0094] The term "antitumor activity" refers to the reduction of tumor cell proliferation, survival rate, or metastatic activity.For example, antitumor activity can be shown by the reduction of the proliferation rate of abnormal cells occurring during the course of therapy, or the stable or reduced size of tumor, or longer survival, as compared with a control without therapy.Such activity can be evaluated using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, mouse mammary tumor virus (MMTV) models, and other models known in the art for investigating antitumor activity.

[0088]

[0095] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the rate of onset or occurrence of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0089]

[0096] The term "CLDN18.2" refers to Claudin-18 splice variant 2 from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.2 is human CLDN18.2. Exemplary sequences of human CLDN18.2 include human CLDN18.2 protein (NCBI reference sequence NP_001002026.1, or SEQ ID NO: 30). Exemplary sequences of CLDN18.2 include Mus musculus (mouse) CLDN18.2 protein (NCBI reference sequence NP_001181852.1) Macaca fascicularis (club-eating monkey) CLDN18.2 protein (NCBI reference sequence XP_015300615.1). CLDN18.2 is expressed in cancer cells. In one embodiment, said CLDN18.2 is expressed on the surface of a cancer cell.

[0090]

[0097] The term "CLDN18.1" refers to Claudin-18 splice variant 1 from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.1 is human CLDN18.1. Exemplary sequences of human CLDN18.1 include human CLDN18.1 protein (NCBI reference sequence number NP_057453.1, or sequence number 31), Mus musculus (mouse) CLDN18.2 protein (NCBI reference sequence number NP_001181851.1), and Macaca fascicularis (crab-eating monkey) CLDN18.2 protein (NCBI reference sequence number XP_005545920.1).

[0091]

[0098] As used herein, a "CLDN18.2-associated" disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise related to increased or decreased expression or activity of CLDN18.2. In some embodiments, the CLDN18.2-associated condition is, for example, cancer.

[0092]

[0099] As used herein, "cancer" refers to any medical condition characterized by malignant cell proliferation or neoplasia, abnormal growth, invasion or metastasis, and includes both solid tumors and non-solid cancers such as leukemia (e.g., hematological malignancies). As used herein, a "solid tumor" refers to a solid mass of neoplastic and / or malignant cells.

[0093]

[0100] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation and physiologically compatible with the recipient thereof.

[0094]

[0101] Reference herein to a value or parameter of "about" includes (and describes) an embodiment that is directed to the value or parameter itself. For example, a reference to "about X" includes the reference to "X". A numerical range includes the numbers defining the range. Generally speaking, the term "about" refers to the stated value of the variable and all values ​​of the variable that are within experimental error of the stated value (e.g., within a 95% confidence interval for the mean) or within 10 percent of the stated value, whichever is greater. When the term "about" is used within the context of a period of time (years, months, weeks, days, etc.), the term "about" means the period plus or minus a certain amount of the next subperiod (e.g., about 1 year means 11-13 months; about 6 months means 6 months ± 1 week; about 1 week means 6-8 days; etc.), or within 10 percent of the stated value, whichever is longer.

[0095]

[0102] Anti-CLDN18.2 antibody conjugate

[0103] The present disclosure provides radionuclide conjugates of anti-CLDN18.2 antibodies.

[0096]

[0104] Claudin-18 (CLDN18) molecules (Genbank accession numbers: splice variant 1 (CLDN18A1 or CLDN18.1): NP_057453, NM_016369, and splice variant 2 (CLDN18A2 or CLDN18.2): NM_001002026, NP_001002026) are integral transmembrane proteins with molecular weights of approximately 27.9 / 27.72 kD. CLDN18 proteins are located within epithelial and endothelial tight junctions, which organize a network of interconnected strands of intramembrane particles between adjacent cells. CLDN18 and occludin are the most prominent transmembrane protein components in tight junctions. Due to their strong cell-cell adhesion properties, these tight junction proteins create a primary barrier to prevent and control paracellular transport of solutes and also limit the lateral diffusion of membrane lipids and proteins, thus maintaining cell polarity.

[0097]

[0105] CLDN18 exhibits several different conformations that can be selectively processed by antibodies (see Sahin U, Koslowski M, Dhaene K, et al. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development[J]. Clinical Cancer Research, 2008, 14(23):7624-7634). CLDN18 conformation 1 has all four hydrophobic regions that serve as transmembrane domains (TM), as described for the majority of CLDN family members, and two extracellular loops are formed (loop 1 encompassed by hydrophobic regions 1 and 2; loop 2 encompassed by hydrophobic regions 3 and 4). The second conformation (CLDN18 conformation 2) entails that the portion between the first and fourth transmembrane domains (loop D3) is extracellular, since the second and third hydrophobic domains do not completely cross the plasma membrane, as described for PMP22. The third conformation (CLDN18 conformation 3) displays a large extracellular domain with two internal hydrophobic regions encompassed by the first and fourth hydrophobic regions. Due to a classical N-glycosylation site in loop D3, the CLDN-18 topology variants CLDN18 topology 2 and CLDN18 topology 3 have additional internal extracellular N-glycosylation sites.

[0098]

[0106] CLDN18 has two distinct splice variants present in both mice and humans. The splice variants CLDN18.1 and CLDN18.2 differ in the first 21 amino acids at the N-terminus, including the first TM and loop 1, whereas the C-terminal protein sequences are identical (see Niimi T, Nagashima K, Ward JM, et al. Claudin-18, a novel downstream target gene for the T / EBP / NKX2.1 homeodomain transcription factor, encodes lung-and stomach-specific isoforms through alternative splicing [J]. Molecular and cellular biology, 2001, 21(21):7380-7390).

[0099]

[0107] CLDN18.1 is selectively expressed in normal lung and gastric epithelium, whereas CLDN18.2 is expressed exclusively in gastric cells. Most importantly, CLDN18.2 expression is restricted to differentiated, short-lived cells of the gastric epithelium, but absent from the gastric stem cell region. Both variants are completely undetectable in other normal human organs, even when using highly sensitive RT-PCR. However, both variants are highly expressed in several cancer types, including gastric, esophageal, pancreatic and lung tumors, as well as in human cancer cell lines (see Matsuda Y, Semba S, Ueda J, et al. Gastric and intestinal claudin expression at the invasive front of gastric carcinoma[J].Cancer science, 2007, 98(7):1014-1019.).

[0100]

[0108] Without wishing to be bound by any theory, it is believed that the molecular and functional characteristics of CLDN18.2 make it a highly interesting target for antibody-based cancer diagnosis and therapy, including (i) the absence of CLDN18 from the majority of toxicity-related normal tissues, (ii) the restriction of CLDN18.2 variant expression to unwanted cell populations such as differentiated gastric cells that can be replenished by gastric target-negative stem cells, (iii) possible differential glycosylation between normal and neoplastic cells, and (iv) the presence of various conformational topologies.

[0101]

[0109] It has been found that the molecular weight of CLDN18 protein differs between tumors and adjacent normal tissues. Higher molecular weight CLDN18 protein is observed in healthy tissues, which can be reduced to the same molecular weight observed in tumors by treatment of normal tissue lysates with the deglycosylating compound PNGase F. This suggests that CLDN18 is less glycosylated in tumors compared to its normal tissue counterparts. The classical N-glycosylation motif is at amino acid residue 116 in the loop D3 domain of the CLDN18 molecule. The molecular weight difference and the predicted structural difference may represent an altered epitope for antibody binding.

[0102]

[0110] In addition, CLDN18 as a tight junction protein can contribute to good specificity for diagnosis and good therapeutic window for treatment. Because tumor cells express CLDN but often do not form classical tight junctions by homotypic and heterotypic association of CLDN as found in normal epithelial tissues, tumor cells are likely to have a significant pool of free CLDN amenable to extracellular antibody binding and immunotherapy. The epitope binding of CLDN in healthy epithelium may be blocked from access to antibody binding within tight junctions.

[0103]

[0111] The present disclosure provides an anti-CLDN18.2 antibody conjugate comprising an anti-CLDN18.2 antibody, or an antigen-binding fragment thereof, conjugated to a radionuclide.

[0104]

[0112] i. Radionuclides

[0113] A radionuclide is an agent characterized by an unstable atomic nucleus that can undergo radioactive decay. Radionuclides useful within the present disclosure include gamma ray emitters, positron emitters, Auger electron emitters, X-ray emitters and fluorescent emitters. Radionuclides can be either therapeutic or diagnostic.

[0105]

[0114] In certain embodiments, the radionuclide is therapeutic. Radionuclides that decay by short-range high-LET emission, such as beta, alpha, and Auger electrons, are highly cytotoxic and therefore useful for labeling materials designed for radiotherapy purposes. Therapeutic radionuclides can have decay energies in the range of 20-6,000 keV, e.g., 60-200 keV for Auger emitters, 100-2,500 keV for beta emitters, and 4,000-6,000 keV for alpha emitters. One of skill in the art can select the appropriate therapeutic nuclide for use in the antibody conjugates provided herein based on factors including the half-life of the radionuclide, the energy of the emitted particles, the maximum range the emitted particles can travel, sufficient concentration and long-term retention of the radionuclide by cancer cells, among others.

[0106]

[0115] Examples of therapeutic radionuclides include, but are not limited to, 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I,32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Examples include Pb.

[0107]

[0116] Examples of additional therapeutic radionuclides that substantially decay into Auger-emitting particles include Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m, and Ir-192.

[0108]

[0117] Examples of further additional radionuclides that substantially decay with the production of alpha particles include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, and Fm-255.

[0109]

[0118] Additional potential therapeutic radionuclides include: 11 C. 13 N, 15 O. 75 Br, 198 Au,224 Ac, 126 I, 133 I, 77 Br, 113m In, 95 Ru, 97 Ru, 103 Ru, 105 Ru, 107 Hg, 203 Hg, 121m Te, 122m Te, 125m Te, 165 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 105 Rh, 142 Pr, 143 Pr, 161 Tb, 166 Ho, 199 Au, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 201 Tl, 225 Ac, 76 Br, 169 Yb, etc.

[0110]

[0119] In certain embodiments, the radionuclide is diagnostic.

[0111]

[0120] Diagnostic radionuclides can be used as imaging agents. Imaging agents can show the location of the radionuclide and its adhering substance in cells or tissues of an animal or human subject, or in cells or tissues under in vitro conditions. In certain embodiments, the radionuclide is one that can be detected externally in a non-invasive manner after administration in vivo. Radionuclides for diagnostic applications, such as imaging agents, preferably have relatively low cytotoxicity, but decay with an emission suitable for imaging. Radionuclides with diagnostic applications include, for example, 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe,59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 l, 124 l, 125 l, 131 l, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154’’1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 lr, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Paramagnetic ions that may be used as diagnostic agents according to embodiments of the present disclosure include, but are not limited to, ions of transition metals and lanthanide metals (e.g., metals having atomic numbers of 6-9, 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

[0112]

[0121] In certain embodiments, the radionuclides are radioactive metal ions, gamma-emitting radioactive halogens and positron-emitting radioactive nonmetals. In certain embodiments, the radionuclides are detectable by positron emission tomography (PET) or single photon emission computed tomography (SPECT). For example, radionuclides that decay with gamma emission are suitable for planar imaging and single photon emission computed tomography (SPECT) imaging, while radionuclides that decay with positron (beta) emission and annihilation photons are suitable for positron emission tomography (PET). Diagnostic radionuclide labels detectable by PET or SPECT imaging techniques include, but are not limited to, for example: 64 Cu, 67 Cu, 89 Zr, 124 I, 86 Y, 90 Y, 111 In, 123 / 131 I, 177 Lu, 11 C. 14 C. 41 Ca, 67 Ga, 68 Ga, 13 N, 15 O. 44 Sc, 18 F, 99m Tc, and 90m Tc, etc.

[0113]

[0122] The term "positron emission tomography (PET)" as used herein refers to a nuclear imaging technique used in the medical field to visualize targeted tissues or organs or activities. PET measures two annihilation photons produced in quick succession after positron emission from a radionuclide-conjugated tracer molecule selected to mark a specific function in the body at the biochemical level. PET provides molecular imaging of biological functions instead of anatomical findings. PET allows for patient examination by creating images of many functions of the human body that are not accessible by other imaging techniques. After being injected into a subject, short-lived positron-emitting radioactive tracers can be distributed in the body following physiological pathways associated with their stable counterparts. When the tracer is a targeting molecule specifically directed to a target of interest, the tracer allows for visualization of tissues or organs expressing such targets.

[0114]

[0123] The term "SPECT" as used herein refers to "single photon emission computed tomography", which is a nuclear medicine tomography imaging technique that uses gamma rays. It is similar to conventional nuclear medicine planar imaging that uses a gamma camera and can provide true 3D information. This information is usually displayed as a cross-section through the patient, but can be freely reformatted or manipulated as needed. Injection of a gamma-emitting radionuclide or its conjugate into the subject is required for imaging.

[0115]

[0124] Methods for producing radionuclide conjugate antibodies and antigen-binding fragments are known in the art. Generally, the methods are different for metallic radionuclides and non-metallic radionuclides. Conjugation of metallic radionuclides usually requires a chelating agent for conjugation, but this chelating agent may not be required for non-metallic radionuclides.

[0116]

[0125] Methods for conjugating metal radionuclides to antibodies, for example, via suitable chelating agents, are known in the art (see, e.g., WO 94 / 11026; Current Protocols in Immunology, Vols. 1 and 2, Coligen et al., eds. Wiley-Interscience, New York, NY, Pubs. (1991)).

[0117]

[0126] The term "chelating agent" as used herein refers to a chemical structure that can bind metals with two or more bonds. A chelating agent can have one or more chelating groups that can bind to metal ions. For example, a chelating agent can contain at least one heteroatom suitable for coordination to a metal ion and sequester the metal ion from an aqueous solution.

[0118]

[0127] In some embodiments, the chelator comprises three or more atoms for chelation, each atom selected from the group consisting of nitrogen, sulfur, oxygen, and phosphorus.

[0119]

[0128] Examples of chelating agents that may be used in accordance with the present disclosure include, but are not limited to, DFO (delferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (NR-diethylenetriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-acetic acid), TRITA (1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid); TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid); and HETA (1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid). ), NETA ({4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), TACN-TM (N,N',N", tris(2-mercaptoethyl) 1,4,7-triazacyclononane), TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]), CP256, PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid), porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and derivatives thereof.

[0120]

[0129] In some embodiments, the antibody conjugate provided herein comprises a metal radionuclide conjugated to the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein via a chelator.Those skilled in the art can select a suitable chelator for the radionuclide based on knowledge known in the art, for example, as described by Price E. W et al., Chem. Soc. Rev., 2014, 43, 260-290.

[0121]

[0130] In some embodiments, the metal radionuclide is 64 Cu, 67 Cu, 89 Zr, 86 Y, 90Y, 111 In, 177 Lu, 67 Ga, 44 Sc, or 99m In some embodiments, the radionuclide may be Tc. 64 Cu, 67 Cu, or 89 In some embodiments, 64 Cu, 67 Cu, or 89 Zr is labeled to the antibody or its antigen-binding fragment via a chelating agent.

[0122]

[0131] In some embodiments, the chelator further comprises a reactive functional group for conjugation to an antibody. The chelator can be directly linked to an antibody or peptide, for example, as disclosed in U.S. Pat. No. 4,824,659, the entire disclosure of which is incorporated herein by reference. Such reactive group allows the chelator to react with one or more amino acid residues of the antibody, such as free cysteine, lysine, and other functional groups. Examples of reactive groups include maleimide, aminobenzyl, N-hydroxysuccinimide ester, and the like.

[0123]

[0132] In certain embodiments, the chelator is conjugated to the antibody or antigen-binding fragment thereof provided herein by a bifunctional linker reagent. Examples of such bifunctional linkers include, but are not limited to, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazo Examples of linker reagents include nium derivatives (e.g., bis-(p-diazonium benzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), bis-active fluorine compounds (e.g., 1,5-difluom-2,4-dinitrobenzene), BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPRH, SBAP, SIA, SIAB, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSG (succinimidyl-(4-vinylsulfone)benzoate). These linker reagents are commercially available (e.g., Pierce Biotechnology, Inc., Rockford, Ill., USA, see pp. 467-498, 2003-2004 Applications Handbook and Catalog).

[0124]

[0133] In some embodiments, the radionuclide is 64 Cu or 67 Cu and the chelator comprises TETA, NOTA, NODA, or NODGA. 89 Zr and the chelating agent comprises DFO.

[0125]

[0134] In some embodiments, the radionuclide is a non-metal. In some embodiments, the radionuclide comprises a radioactive halogen. In some embodiments, the radionuclide is 124 I or 123 I or 131 I. Methods for preparing radioactive iodinated antibodies are known in the art, see, for example, Grassi, J. et al. (1987). Radioiodination and Other Labeling Techniques. Handbook of Experimental Pharmacology, pp. 91-141; Eclelman, WC et al. Cancer Research, 40, pp. 3036-3042, 1998, which are incorporated herein by reference in their entirety.

[0126]

[0135] In some embodiments, 124 I or 123 I or 131 I is labeled at least one phenyl hydroxyl group of the antibody or antigen-binding fragment thereof.

[0127]

[0136] In certain embodiments, the antibody or antigen-binding fragment thereof is 124 The antibody or antigen-binding fragment thereof is iodized directly by electrophilic substitution at the tyrosine residue in the presence of a radioactive iodine such as I. In certain embodiments, the antibody or antigen-binding fragment thereof is indirectly iodized by covalent attachment of a pre-labeled compound or a compound that can be labeled after attachment, e.g., by Bolton-Hunter reagent for peptides.

[0128]

[0137] In some embodiments, 124 I or 123 I or 131 I is labeled to antibody or its antigen-binding fragment in the presence of oxidizing agent.Any suitable oxidizing agent can be used, including chemical oxidizing agent and enzymatic oxidizing agent.Exemplary oxidizing agents include, but are not limited to, N-bromosuccinimide, chloramine T, chlorine gas, or lactoperoxidase.

[0129]

[0138] In some embodiments, 124 I or 123 I or 131 I is labeled onto an antibody or an antigen-binding fragment thereof via N-bromosuccinimide (NBS) reaction.

[0130]

[0139] In some embodiments, the antibody conjugate provided herein can be further conjugated to nanoparticles.For example, nanoparticles can be used in therapeutic applications as drug carriers, which, when conjugated to the CLDN18.2-specific antibody or fragment of the present invention, deliver chemotherapeutic agents, radiotherapeutic agents, toxins, or any other cytotoxic or anticancer agents known in the art to cancerous cells that overexpress CLDN18.2 on the cell surface.

[0131]

[0140] In some embodiments, the antibody conjugates provided herein can be further conjugated to a drug (eg, at the epsilon amino group of a lysine residue), while the carrier can incorporate an additional therapeutic or diagnostic agent.

[0132]

[0141] i. Antibody sequence

[0142] In certain embodiments, the antibody conjugates provided herein comprise an anti-CLDN18.2 antibody or antigen-binding fragment thereof comprising the sequences of heavy chain HCDR1, HCDR2, and HCDR3, and / or light chain LCDR1, LCDR2, and LCDR3, wherein: the HCDR1 sequence comprises GYNMN (SEQ ID NO:1), or TYFIGVG (SEQ ID NO:13), or a homologous sequence thereof with at least 80% sequence identity; the HCDR2 sequence comprises X1IDPYYX2X3TX4YNQKFX5G (SEQ ID NO: 32), or HIWWNDNKYYNTALKS (SEQ ID NO: 15), or a homologous sequence thereof with at least 80% (or at least 85%, 90%, 95%) sequence identity; the HCDR3 sequence comprises X6X7X8GNAFDY (SEQ ID NO:33), or MGSGAWFTY (SEQ ID NO:17), or a homologous sequence of at least 80% sequence identity thereof; The LCDR1 sequence is KSSQX9LX 10 NX 11 GNX 12 KNYLT (SEQ ID NO:34), or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto; LCDR2 array is WASTRX 13 S (SEQ ID NO:35), or a homologous sequence having at least 80% sequence identity thereto; LCDR3 array is QNDYX 14 X 15 PX 16 T (SEQ ID NO: 36), or a homologous sequence having at least 80% sequence identity thereto; where X1 is N or Y or H, X2 is G or V, X3 is A or G or T, X4 is R or T or S, X5 is K or R, X6 is S or M, X7 is Y or F, X8 is Y or H, X9 is S or N, and X 10 is L or F, and X 11 is S or N, and X 12 is Q or L, and X 13 is E or K, and X 14 is S or Y, and X 15 is F or Y and X 16 is F or L.

[0133]

[0143] In certain embodiments, the antibody conjugate provided herein comprises an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region is: a) HCDR1 comprising a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 13, b) HCDR2 comprising a sequence selected from SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:19, and SEQ ID NO:22, and c) HCDR3 comprising a sequence selected from SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, and SEQ ID NO:21; and / or The light chain variable region is: d) LCDR1 comprising the sequence of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:20; e) LCDR2 comprising the sequence of SEQ ID NO: 4 and SEQ ID NO: 16, and f) LCDR3 comprising a sequence selected from SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:18. Includes.

[0134]

[0144] In certain embodiments, the heavy chain variable region is selected from the group consisting of: a) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:3, and an HCDR3 comprising the sequence of SEQ ID NO:5; b) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:7, and an HCDR3 comprising the sequence of SEQ ID NO:5; c) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:9, and an HCDR3 comprising the sequence of SEQ ID NO:11; d) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 15, and an HCDR3 comprising the sequence of SEQ ID NO: 17; e) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:19, and an HCDR3 comprising the sequence of SEQ ID NO:21; and f) A heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:22, and an HCDR3 comprising the sequence of SEQ ID NO:5.

[0135]

[0145] In certain embodiments, the light chain variable region is selected from the group consisting of: a) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; b) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:8; c) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; d) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:12; e) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 14, an LCDR2 comprising the sequence of SEQ ID NO: 16, and an LCDR3 comprising the sequence of SEQ ID NO: 18; and f) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:20, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6.

[0136]

[0146] In certain embodiments, in the antibody conjugates provided herein: a) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:3, and an HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; b) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:7, and an HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:8; c) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:9, and an HCDR3 comprising the sequence of SEQ ID NO:11; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:10, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; d) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 15, and an HCDR3 comprising the sequence of SEQ ID NO: 17; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 12; e) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:19, and an HCDR3 comprising the sequence of SEQ ID NO:21; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:16, and an LCDR3 comprising the sequence of SEQ ID NO:18; or f) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:22, and an HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:20, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6.

[0137]

[0147] In certain embodiments, the antibody conjugates provided herein comprise one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of CLDN18.2 antibodies 7C12, 11F12, 26G6, 59A9, 18B10, and 12E9.

[0138]

[0148] As used herein, "7C12" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:37 and a light chain variable region of SEQ ID NO:38.

[0139]

[0149] As used herein, "11F12" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:39 and a light chain variable region of SEQ ID NO:40.

[0140]

[0150] As used herein, "26G6" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:41 and a light chain variable region of SEQ ID NO:42.

[0141]

[0151] As used herein, "59A9" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:43 and a light chain variable region of SEQ ID NO:44.

[0142]

[0152] As used herein, "18B10" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:45 and a light chain variable region of SEQ ID NO:46.

[0143]

[0153] As used herein, "12E9" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:47 and a light chain variable region of SEQ ID NO:48.

[0144]

[0154] Table 1 shows the CDR sequences of these CLDN18.2 antibodies. The heavy and light chain variable region sequences are also provided below in Table 2.

[0145]

[0155]

[0146] [Table 1]

[0147]

[0156]

[0148] [Table 2]

[0149]

[0157] The anti-CLDN18.2 antibodies or antigen-binding fragments thereof provided herein can be monoclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, bivalent antibodies, or anti-idiotypic antibodies. Recombinant antibodies are antibodies prepared in vitro using recombinant methods rather than in an animal.

[0150]

[0158] CDRs are known to be responsible for antigen binding, but it has been found that not all six CDRs are necessarily essential or invariant. In other words, it is possible to replace or change or modify one, two or three CDRs in anti-CLDN18.2 antibodies 7C12, 11F12, 26G6, 59A9, 18B10 or 12E9 (corresponding to any one of SEQ ID NOs: 1-22) and still substantially retain the specific binding affinity to CLDN18.2.

[0151]

[0159] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein comprise the heavy chain CDR3 sequence of one of the anti-CLDN18.2 antibodies 7C12, 11F12, 26G6, 59A9, 18B10, or 12E9. In certain embodiments, the antibody conjugates provided herein comprise the heavy chain CDR3 sequences of SEQ ID NOs: 5, 11, 17, and 21. The heavy chain CDR3 region is located at the center of the antigen-binding site and is therefore believed to make the most contacts with the antigen and provide the most free energy for the affinity of the antibody to the antigen. The heavy chain CDR3 is also believed to be by far the most diverse CDR for the antigen-binding site in terms of length, amino acid composition, and conformation due to multiple diversification mechanisms (Tonegawa S. Nature. 302:575-81). Diversity in the heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45) as well as desirable antigen binding affinities (Schier R, et al. J Mol Biol. 263:551-67).

[0152]

[0160] In some embodiments, the anti-CLDN18.2 antibody conjugates provided herein comprise all or part of the heavy chain variable domain and / or all or part of the light chain variable domain. In one embodiment, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein are single-domain antibodies consisting of all or part of the heavy chain variable domain provided herein. More information regarding such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0153]

[0161] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof can specifically bind to CLDN18.2. The CDR sequences provided in Table 1 are obtained from mouse antibodies, but can be grafted to any suitable FR sequence of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant techniques.

[0154]

[0162] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein are humanized. Humanized antibodies or antigen-binding fragments are desirable in terms of their reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be essentially performed by substituting non-human (e.g., mouse) CDR genes for the corresponding human CDR genes in human immunoglobulin genes (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0155]

[0163] Suitable human heavy and light chain variable domains can be selected to this end using methods known in the art. In an illustrative example, a "best-fit" approach can be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain germline sequences, and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J.Mot.Biol. 196:901). Alternatively, frameworks derived from the consensus sequence of all human antibodies can be used for grafting of non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0156]

[0164] In certain embodiments, the humanized antibody conjugates provided herein are composed of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FR, and, if present, the constant region, are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences may be derived from different human immunoglobulin genes, e.g., the FR sequences may be derived from one human antibody and the constant region may be derived from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment comprises human heavy / light chain FR1-4.

[0157]

[0165] In some embodiments, the FR region derived from a human may contain the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are replaced with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or fragment thereof more similar to the non-human parent antibody structure, reducing or avoiding immunogenicity, and / or improving or retaining binding activity or binding affinity.

[0158]

[0166] In certain embodiments, the humanized antibody conjugates provided herein contain no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each human FR sequence, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs of the heavy or light chain variable domain. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. In certain embodiments, one or more amino acid residues are mutated, for example, back-mutated to the corresponding residue found in the non-human parent antibody from which the CDR sequence is derived (e.g., in the mouse framework region). The positions suitable for mutation can be selected by the skilled artisan according to principles known in the art. For example, the position of the mutation may be selected at the following locations: 1) where the residue in the framework of the human Gremlin sequence is rarely present (e.g., in less than 20% or less than 10% of the human variable region sequences); 2) where the location is directly adjacent to one or more of the three CDRs in the primary sequence of the human germline chain, because it is more likely to interact with the residues in the CDRs; or 3) where the location is close to the CDRs in the three-dimensional model, and therefore may have a good possibility of interacting with the amino acids in the CDRs. The residue at the selected location may be backmutated to the corresponding residue in the parent antibody, or to a residue that is neither the corresponding residue in the human germline sequence nor the corresponding residue in the parent antibody, but is a common residue in human sequences, i.e., occurs more frequently at that position in known human sequences that belong to the same subgroup as the human germline sequence (see U.S. Patent No. 5,693,762).

[0159]

[0167] In certain embodiments, the humanized light chains and humanized heavy chains of the present disclosure are substantially non-immunogenic in humans and retain substantially the same, or even higher, affinity for CLDN18.2 as the parent antibody.

[0160]

[0168] In certain embodiments, the humanized antibody conjugates provided herein comprise one or more light chain FR sequences of human germline framework sequence VK / 4-1, with or without back mutations, and / or one or more heavy chain FR sequences of human germline framework sequence VH / 1-46. Back mutations may be introduced into the human germline framework sequences, if desired. In certain embodiments, the humanized antibody 18B10 may contain one or more back mutations in the heavy chain framework sequence VH / 1-46 selected from the group consisting of R71I, T73K, T28S, M69L, R38K, and M48I, all based on the Kabat numbering. The humanized antibody 18B10 may contain one or more back mutations in the light chain framework sequence VK / 4-1 selected from the group consisting of S63T and I21M, all based on the Kabat numbering.

[0161]

[0169] In certain embodiments, in the anti-CLDN18.2 antibody conjugates provided herein, the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding affinity to CLDN18.2, in particular human CLDN18.2.

[0162]

[0170] In certain embodiments, in the anti-CLDN18.2 antibody conjugates provided herein, the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding affinity to CLDN18.2, particularly human CLDN18.2.

[0163]

[0171] In certain embodiments, the anti-CLDN18.2 antibody conjugate provided herein comprises: a heavy chain variable region comprising the sequence of SEQ ID NO:25 and a light chain variable region comprising the sequence of SEQ ID NO:26; a heavy chain variable region comprising the sequence of SEQ ID NO:27 and a light chain variable region comprising the sequence of SEQ ID NO:28; a heavy chain variable region comprising the sequence of SEQ ID NO: 29 and a light chain variable region comprising the sequence of SEQ ID NO: 26 or 28; a heavy chain variable region comprising the sequence of SEQ ID NO: 37 and a light chain variable region comprising the sequence of SEQ ID NO: 38; A heavy chain variable region comprising the sequence of SEQ ID NO: 39 and a light chain variable region comprising the sequence of SEQ ID NO: 40; a heavy chain variable region comprising the sequence of SEQ ID NO: 41 and a light chain variable region comprising the sequence of SEQ ID NO: 42; a heavy chain variable region comprising the sequence of SEQ ID NO: 43 and a light chain variable region comprising the sequence of SEQ ID NO: 44; A heavy chain variable region comprising the sequence of SEQ ID NO: 45 and a light chain variable region comprising the sequence of SEQ ID NO: 46; or A heavy chain variable region comprising the sequence of SEQ ID NO:47 and a light chain variable region comprising the sequence of SEQ ID NO:48.

[0164]

[0172] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein further comprise one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4, in which: HFR1 is QVQLVQSGAEVKKPGASVKVSCKASGYX 17 FT (SEQ ID NO:54) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto; HFR2 is WVX 18 QAPGQGLEWX 19 G (SEQ ID NO:55) or a homologous sequence having at least 80% (or at least 90%) sequence identity thereto; The HFR3 sequence is RVTX 20 TIDKSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO:56), or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto; HFR4 comprises WGQGTTVTVSS (SEQ ID NO:57) or a homologous sequence thereof of at least 80% sequence identity; LFR1 is DIVMTQSPDSLAVSLGERATX 21 NC (SEQ ID NO:58) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto; LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO:59) or a homologous sequence thereof of at least 80% (or at least 85%, 90%) sequence identity; LFR3 is GVPDRFX 22 GSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 60) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto; LFR4 comprises FGGGTKVEIK (SEQ ID NO: 61) or a homologous sequence thereof with at least 80% (or at least 90%) sequence identity; Where X 17is T or S, and X 18 is R or K, and X 19 is M or I, and X 20 is M or L, and X 21 is I or M, and X 22 is S or T.

[0165]

[0173] In certain embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 62 and 63, HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 64 and 65, HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 66 and 67, HFR4 comprises the sequence of SEQ ID NO: 57, LFR1 comprises a sequence from the group consisting of SEQ ID NOs: 68 and 69, LFR2 comprises the sequence of SEQ ID NO: 59, LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 70 and 71, and LFR4 comprises the sequence of SEQ ID NO: 61.

[0166]

[0174]

[0167] [Table 3]

[0168]

[0175] Table 3-2 illustrates the sequences of the variable regions of the humanized 18B10 antibody.

[0169]

[0176]

[0170] [Table 4-1]

[0171] [Table 4-2]

[0172]

[0177] In certain embodiments, the humanized antibodies provided herein can comprise a heavy chain variable region fused to a constant region of a human IgG1 isotype and a light chain variable region fused to a constant region of a human kappa chain.

[0173]

[0178] The humanized anti-CLDN18.2 antibody conjugates provided herein retained specific binding affinity to CLDN18.2-expressing cells, at least comparable to or even better than the parent antibody in these respects. The humanized antibodies provided herein can also retain their functional interaction with CLDN18.2-expressing cells, such as NUGC4 cells, SNU-620 cells, SNU-601 cells, or KATOIII cells, in that all humanized antibodies provided herein can mediate cell killing by ADCC, CDC, and induction of apoptosis induced by cross-linking of targets at the tumor cell surface, as well as direct inhibition of proliferation. In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region, hi certain embodiments, the light chain constant region comprises a Cκ or Cλ region.

[0174]

[0179] In certain embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein further comprise a constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein comprise a constant region of an IgG1 isotype. In certain embodiments, the constant region of human IgG1 comprises SEQ ID NO: 49, or a homologous sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto.

[0175]

[0180] The constant region of IgG1 isotype can induce effector function such as ADCC or CDC. The effector function of the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein can cause cytotoxicity against cells expressing CLDN18.2. Effector function can be evaluated using various assays, such as Fc receptor binding assay, C1q binding assay, and cytolysis assay, and any of the above assays for determining ADCC or CDC.

[0176]

[0181] Surprisingly, the present inventors have found that the anti-CLDN18.2 antibodies and fragments thereof provided herein have high affinity to cell lines that moderately express human CLDN18.2 (e.g., NUGC4 cells), and cell lines that lowly express human CLDN18.2 (e.g., SNU-620, SNU-601, and KATOIII cells). This is distinguished from existing antibodies such as IMAB362, which do not show specific or comparable binding to cells that lowly express human CLDN18.2. The chimeric IgG1 antibody IMAB362 is an anti-human CLDN18.2 antibody developed by Ganymed Pharmaceuticals AG, having the amino acid sequence disclosed in US Patent Application No. 2009169547 A1 (the variable region sequences of the heavy and light chains of IMB362 are included herein as SEQ ID NO: 72 and SEQ ID NO: 73) and CAS number 1496553-00-4. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 and does not bind to any other claudin family members, including the closely related splice variant 1 of claudin 18 (CLDN18.1).

[0177]

[0182] NUGC4 cells are a cell line established from a paragastric lymph node derived from a cancer patient (see Akiyama S et al., Jpn J Surg. July 1988;18(4):438-46). The NUGC4 cell line is available from the JCRB Cell Bank under the accession number JCRB0834.

[0178]

[0183] Both SNU-601 and SNU-620 cells are human gastric cancer cell lines established by Seoul National University (SNU) from ascites of cancer patients (KU JL et al., Cancer Res Treat. 2005 Feb;37(1):1-19; Park et al., Int J Cancer. 1997 Feb;70(4):443-449). SNU-601 and SNU-620 cells are available from the Korean Cell Line Bank under accession numbers 00601 and 00620, respectively.

[0179]

[0184] KATO III cells are a cell line derived from a metastasis of a gastric cancer patient (see Sekiguchi M, et al. Jpn. J. Exp. Med. 48:61-68, 1978.) The KATO III cell line is available from the ATCC under the accession number ATCC HTB-103.

[0180]

[0185] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein have a K of 2.5 nM or less (or 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or less) as measured by a KinExA assay. D In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein specifically bind to human CLDN18.2-expressing cells (e.g., NUGC4 or KATOIII cell lines) with a Kd value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or less of the Kd value of IMAB362 as measured by KinExA assay. In certain embodiments, the Kd value is DThe value is determined using NUGC4 cells, KATOIII cells, SNU-601 cells, SNU-620 cells, or equivalent cells thereof having human CLDN18.2 protein expression levels comparable to or lower than those of NUGC4 cells, KATOIII cells, SNU-601 cells, or SNU-620 cells. In certain embodiments, K D Values ​​are determined using cell lines that highly express human CLDN18.2 or that moderately express human CLDN18.2.

[0181]

[0186] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein have an EC50 value of 70 μg / ml or less (or 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / ml or less) for binding to human CLDN18.2 (or murine CLDN18.2)-expressing cells, as measured by flow cytometry assay. In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein specifically bind to human CLDN18.2-expressing cells with an EC50 value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 1%, or 0.1% or less of the EC50 value of IMAB362 as measured by flow cytometry assay. In certain embodiments, EC50 value is determined using NUGC4 cell line, KATOIII cell line, SNU-601 cell line, SNU-620 cell line, or equivalent cells thereof, which have human CLDN18.2 protein expression levels comparable to or lower than those of NUGC4 cell line, KATOIII cell line, SNU-601 cell line, or SNU-620 cell line, for example, using a cell line that expresses human CLDN18.2 at low or medium levels.In certain embodiments, EC50 is determined using a cell line that expresses human CLDN18.2 at high levels.

[0182]

[0187] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein have EC50 values ​​of 5, 4, 3, or 2 μg / ml or less for binding to a cell line that highly expresses human CLDN18.2 or a cell line that moderately expresses human CLDN18.2.

[0183]

[0188] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein have an EC50 value for binding to NUGC4 cells of 70 μg / ml or less (or 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / ml or less) as measured by flow cytometry assay.

[0184]

[0189] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein do not bind to CLDN18.1 (eg, human CLDN18.1 or murine CLDN18.1).

[0185]

[0190] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein can specifically bind to murine CLDN18.2 (e.g., cells expressing murine CLDN18.2) with an EC50 value of 1.5 μg / ml or less as measured by flow cytometry. In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein bind to murine CLDN18.2 with an EC50 of 0.1 μg / ml to 1.5 μg / ml (e.g., 0.1 μg / ml to 1.2 μg / ml, 0.2 μg / ml to 1 μg / ml, 0.5 μg / ml to 1 μg / ml, 0.6 μg / ml to 1 μg / ml, 0.6 μg / ml to 0.8 μg / ml, or 0.67 μg / ml) as measured by flow cytometry.

[0186]

[0191] In certain embodiments, the anti-CLDN18.2 antibodies provided herein and the anti-CLDN18.2 antibody conjugates provided herein are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or CDC activity in cells expressing various levels of human CLDN18.2.

[0187]

[0192] As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. The lysis of target cells is extracellular, requires direct cell-cell contact, and does not involve complement. ADCC can be viewed as a mechanism that directly induces various degrees of immediate tumor destruction, resulting in antigen presentation and induction of tumor-specific T cell responses. In vivo induction of ADCC is believed to result in tumor-specific T cell responses and host-derived antibody responses.

[0188]

[0193] Methods for performing ADCC are known in the art. In general, target cells, such as CLDN18.2-expressing cells, are incubated with a range of concentrations of anti-CLDN18.2 antibodies, and after washing, effector cells, such as Fc receptor-expressing cells, are added to allow ADCC to occur. Cytotoxicity or cell viability is determined at a time point several hours after mixing of the effector and target cells, thereby quantifying the level of ADCC. Cytotoxicity can be detected by the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein such as lactate dehydrogenase (LDH)) from lysed target cells. In another embodiment, cell viability is determined by an indicator of metabolically active cells (e.g., ATP) using a luciferase reporter gene that generates a luminescent signal proportional to the number of live cells in culture (i.e., ADCC reporter assay) (see, e.g., Crouch, SP et al. (1993) J. Immunol. Methods 160, pp. 81-8). Examples of effector cells are NK cells, PBMCs, or FcγRIII expressing cells.

[0189]

[0194] "Complement-dependent cytotoxicity" or "CDC" is another antibody-induced method of cell killing by lysis of the target in the presence of complement. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are also highly effective at inducing CDC via the classical complement activation pathway. In this cascade, formation of an antigen-antibody complex leads to exposure of multiple Clq binding sites in close proximity on the CH2 domain of the participating antibody molecule, such as an IgG molecule complexed with the cognate antigen (Clq is one of the three subcomponents of complement C1). These exposed Clq binding sites convert the previously low affinity Clq-IgG interaction into a high avidity interaction, which sets in motion a cascade of events involving a series of other complement proteins, resulting in the proteolytic release of the effector cell chemotactic / activating agents C3a and C5a. The complement cascade culminates in the formation of the membrane attack complex (MAC), which creates pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0190]

[0195] CDC activity can be determined by a method similar to that of ADCC activity discussed above, except that no effector cells are used, and the presence of complement derived from human serum is required. Briefly, antibody samples are serially diluted in assay medium and incubated with target cells expressing CLDN18.2 in the presence of human serum complement. After incubation, cytotoxicity or cell viability is determined by the release of label from lysed target cells or by an indicator of metabolically active cells (e.g., ATP). CellTiter-Glo reagent, which assays ATP in metabolically active cells, can be used, and the degree of cell lysis can be quantified by measuring the intensity of luminescence using an appropriate reader.

[0191]

[0196] In certain embodiments, ADCC or CDC-induced cell death mediated by the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein can be determined by loss of membrane integrity, as assessed by uptake of propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)), or 7AAD, which can be assessed compared to untreated cells.

[0192]

[0197] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein can induce complement-dependent cytotoxicity (CDC) on cells expressing human CLDN18.2 with an EC50 value of 1 μg / ml or less (or 0.9, 0.8, 0.7, 0.6, 0.5 μg / ml or less) as measured by cytotoxicity assay. In certain embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein can induce CDC on cells expressing human CLDN18.2 with an EC50 value of 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less of the EC50 value of IMAB362 as measured by cytotoxicity assay. In certain embodiments, CDC is determined using a cell line that moderately expresses human CLDN18.2 or a cell line that highly expresses human CLDN18.2.

[0193]

[0198] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein are capable of inducing antibody-dependent cellular cytotoxicity (ADCC) on cells expressing human CLDN18.2 with an EC50 value of 2 μg / ml or less (or 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μg / ml or less) as measured by an ADCC reporter assay. In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein induce ADCC on cells expressing human CLDN18.2 with an EC50 value of 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% or less of the EC50 value of IMAB362, as measured by an ADCC reporter assay, or with a total ADCC potency of at least 120%, 150%, 180%, or 200% of the total ADCC potency of IMAB362 (e.g., as indicated by the maximum level of ADCC activity observed in a plot of antibody concentration versus ADCC activity level). In certain embodiments, ADCC is determined using the NUGC4 cell line, the KATOIII cell line, the SNU-601 cell line, the SNU-620 cell line, or equivalent cells thereof having human CLDN18.2 protein expression levels comparable to or lower than those of the NUGC4 cell line, the KATOIII cell line, the SNU-601 cell line, or the SNU-620 cell line, e.g., a cell line that moderately expresses human CLDN18.2 or a cell line that lowly expresses human CLDN18.2.

[0194]

[0199] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein are capable of inducing ADCC on NUGC4 cells with an EC50 value of 2 μg / ml or less (or 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μg / ml or less) as measured by an ADCC reporter assay.

[0195]

[0200] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein bind to an epitope that includes at least one or more (e.g., 1, 2, 3 or more) of the amino acid residues at positions D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79 and R80 of human CLDN18.2 having the amino acid sequence of SEQ ID NO: 30.

[0196]

[0201] The term "epitope" as used herein refers to a specific group of atoms or amino acids of an antigen to which an antibody binds. An epitope can include specific amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups that directly contact the antibody. Those skilled in the art will understand that it is possible to determine, without undue experimentation, whether an antibody binds to the same epitope as an antibody of the present disclosure (e.g., any of the hybridoma / chimeric or humanized antibodies 7C12, 11F12, 26G6, 59A9, 18B10 provided herein, and their chimeric and humanized variants) or an overlapping epitope or an adjacent epitope by determining whether the two antibodies compete for binding to a CLDN18.2 antigen polypeptide.

[0197]

[0202] The term "compete for binding" as used herein with respect to two antigen-binding proteins (e.g., antibodies) means that one antigen-binding protein blocks or reduces the binding of the other antigen-binding protein to an antigen (e.g., human / mouse CLDN18.2) as determined by competitive binding assays. Competitive binding assays are well known in the art and include, for example, direct or indirect radioimmunoassays (RIA), direct or indirect enzyme immunoassays (EIA), and sandwich competition assays (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253). Typically, such assays involve the use of purified antigen or antigen-bearing cells bound to a solid surface, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess. When two antibodies compete for binding to CLDN18.2, the two antibodies bind to the same or overlapping epitopes, or adjacent epitopes close enough to sterically interfere with the epitope bound by the other antibody. Typically, a competing antibody, when present in excess, will inhibit (e.g., reduce) specific binding of a test antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90% or more.

[0198]

[0203] In certain embodiments, the epitope or amino acid residue of the epitope bound by the antibody can be determined by mutating a specific residue of the antigen, i.e., CLDN18.2. If the antibody binds to mutant CLDN18.2 with an amino acid residue mutated, for example, to alanine, at a significantly reduced level compared to its binding to wild-type CLDN18.2, this indicates that the mutated residue is directly involved in the binding of the antibody to the CLDN18.2 antigen or is in close proximity to the antibody when the antibody is bound to the antigen. Such a mutated residue is considered to be within the epitope, and the antibody is considered to specifically bind to the epitope containing that residue. As used herein, a significantly reduced level of binding means that the binding affinity (e.g., EC50, Kd, ​​or binding ability) between the antibody and mutant CLDN18.2 is reduced by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the binding between the antibody and wild-type CLDN18.2. Such binding measurements can be performed using any suitable method known in the art and disclosed herein, including, but not limited to, KinExA assay and flow cytometry.

[0199]

[0204] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein exhibit significantly lower binding to mutant CLDN18.2 in which the residues of wild-type CLDN18.2 are replaced with alanine, and the residues are selected from the group consisting of D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79, and R80 of human CLDN18.2. In certain embodiments, the residue is E56. In certain embodiments, the residue is selected from the group consisting of W30, L49, W50, R55, and E56. In certain embodiments, the residue is selected from the group consisting of T41, N45, Y46, R51, F60, E62, and R80. In certain embodiments, the residues are selected from the group consisting of D28, V43, N45, Y46, Y66, L72, L76, and V79.

[0200]

[0205] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein exhibit at least 80%, 90%, 95%, or 99% or more reduction in binding to mutant CLDN18.2 containing E56A of human CLDN18.2 compared to binding between the antibody and wild-type CLDN18.2.

[0201]

[0206] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein exhibit at least a 50%, 60%, 70%, 80%, or 90% reduction in binding to mutant CLDN18.2 comprising one or more mutated residues selected from the group consisting of W30A, L49A, W50A, R55A, and E56A of human CLDN18.2, compared to binding between the antibody and wild-type CLDN18.2.

[0202]

[0207] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein exhibit at least a 30%, 35%, 40%, 45%, or 50% reduction in binding to mutant CLDN18.2 comprising one or more mutated residues selected from the group consisting of D28, V43, N45, Y46, Y66, L72, L76, and V79 of human CLDN18.2 compared to binding between the antibody and wild-type CLDN18.2.

[0203]

[0208] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein exhibit at least a 10%, 15%, 20%, 25%, or 30% reduction in binding to mutant CLDN18.2 comprising one or more mutated residues selected from the group consisting of T41A, N45A, Y46A, R51A, F60A, E62A, and R80A of human CLDN18.2 compared to binding between the antibody and wild-type CLDN18.2.

[0204]

[0209] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein do not bind to A42 and / or N45.

[0205]

[0210] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein are capable of binding to epitopes provided herein and of inducing ADCC or CDC activity in cell lines that moderately express human CLDN18.2 or that lowly express human CLDN18.2.

[0206]

[0211] ii. Antibody variants

[0212] The anti-CLDN18.2 antibodies and antigen-binding fragments thereof in the anti-CLDN18.2 antibody conjugates provided herein also encompass various types of variants of the antibody sequences provided herein.

[0207]

[0213] In certain embodiments, the variants contain one or more modifications or substitutions in one, two, or three CDR sequences provided in Table 1, in one or more FR sequences, in the variable region sequences of the heavy or light chains provided herein, and / or in the constant region (e.g., Fc region). Such antibody variants retain the specific binding affinity of their parent antibody to CLDN18.2, but have one or more desirable properties imparted by the modifications or substitutions. For example, the antibody variants may have improved antigen binding affinity, improved glycosylation patterns, reduced risk of glycosylation, reduced deamination, reduced or improved effector functions, improved FcRn receptor binding, extended pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues), to name a few.

[0208]

[0214] The parent antibody sequence can be screened to identify suitable or preferred residues for modification or substitution using methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are generated and screened for the desired properties. If substitution at a particular amino acid position manifests a desired functional change, that position can be identified as a potential residue for modification or substitution. Potential residues can be further evaluated by substituting with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0209] a) Affinity variants

[0215] The affinity variants retain the specific binding affinity of the parent antibody to CLDN18.2, or even have improved specific binding affinity to CLDN18.2 over the parent antibody. Various methods known in the art can be used to achieve this goal. For example, a library of antibody variants (e.g., Fab variants or scFv variants) can be generated and expressed using phage display technology, and then screened for binding affinity to human CLDN18.2. For another example, computer software can be used to virtually simulate the binding of an antibody to human CLDN18.2 and identify amino acid residues on the antibody that form the binding interface. Such residues can be avoided in substitutions to prevent a decrease in binding affinity, or targeted for substitutions that result in stronger binding.

[0210]

[0216] In certain embodiments, at least one (or all) of the substitutions in the CDR sequence, FR sequence, or variable region sequence comprises conservative substitution. "Conservative substitution" in the context of amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitution can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative substitution usually does not cause significant changes in the conformational structure of a protein, and thus may retain the biological activity of the protein.

[0211]

[0217] In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variants comprise a total of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less substitutions in one or more CDR sequences and / or FR sequences.

[0212]

[0218] In certain embodiments, anti-CLDN18.2 antibodies and antigen-binding fragments thereof comprise one, two, or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the CDR sequence(s) listed in Table 1, while retaining binding affinity to CLDN18.2 at a similar or even higher level than its parent antibody.

[0213]

[0219] In certain embodiments, anti-CLDN18.2 antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the variable region sequence(s) of SEQ ID NOs: 23-29 and 37-48, while retaining a binding affinity to CLDN18.2 at a similar or even higher level than that of its parent antibody. In some embodiments, a total of 1-10 amino acids are substituted, inserted, or deleted in a sequence selected from SEQ ID NOs: 25-29 and 37-48. In some embodiments, the substitutions, insertions, or deletions occur in the regions outside the CDRs (i.e., in the FRs).

[0214] b) Glycosylation variants

[0220] The anti-CLDN18.2 antibodies and antigen-binding fragments in the anti-CLDN18.2 antibody conjugates provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.The term "glycosylation" as used herein refers to the enzymatic process of attaching glycans, such as fucose, xylose, mannose, or GlcNAc phosphoserine glycans, to proteins, lipids, or other organic molecules.Depending on the carbon that is linked to the glycan, glycosylation can be divided into five classes, including N-linked glycosylation, O-linked glycosylation, phosphate glycosylation, C-linked glycosylation, and glypiation.

[0215]

[0221] Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in a tripeptide sequence, such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine.

[0216]

[0222] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein include glycosylation variants that have improved effector function, such as ADCC or CDC.

[0217]

[0223] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein is afucosylated. The terms "afucosylation" or "afucosylated" refer to the reduction or removal of core fucose on the N-glycan attached to the antibody. The majority of glycans of human IgG antibodies are known as G0, G1, and G2, which are complex biantennary molecules with 0, 1, or 2 terminal galactose core fucose residues.

[0218]

[0224] Afucosylated antibody variants are described, for example, in US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. They can be made using methods known in the art, such as those described in J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

[0219]

[0225] In certain embodiments, the antibody glycosylation variant is afucosylated at Asn297 site in the CH2 region of the Fc of the antibody. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300.

[0220]

[0226] In certain embodiments, antibody glycosylation variants may be obtained by removal of a native glycosylation site (e.g., by N297A substitution), such that, for example, the tripeptide sequence of the N-linked glycosylation site, or the serine or threonine residue of the O-linked glycosylation site, is no longer present in the antibody or in the Fc sequence. Alternatively, in certain embodiments, antibody glycosylation variants may be obtained by producing the antibody in a host cell line that is not capable of adding the selected sugar group to the mature core carbohydrate structure of the antibody.

[0221] c) Cysteine ​​engineered variants

[0227] The anti-CLDN18.2 antibodies and antigen-binding fragments in the anti-CLDN18.2 antibody conjugates provided herein also encompass cysteine ​​engineered variants, which contain one or more introduced free cysteine ​​amino acid residues.

[0222]

[0228] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bridge.Cysteine ​​engineered variants are useful for, for example, conjugation with cytotoxic compounds and / or imaging compounds, labels, or radioisotopes, among others, at the site of engineered cysteine, for example, through maleimide or haloacetyl.Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0223] d) Fc variants

[0229] The anti-CLDN18.2 antibodies and antigen-binding fragments in the anti-CLDN18.2 antibody conjugates provided herein also encompass Fc variants comprising modification or substitution of one or more amino acid residues in the Fc region and / or hinge region thereof.

[0224]

[0230] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein comprise a constant region comprising one or more amino acid residue substitutions or modifications that confer improved CDC or ADCC compared to the wild-type constant region. Certain amino acid residues in the CH2 domain of the Fc region may be substituted to provide enhanced ADCC activity, for example by increasing the affinity of the Fc domain to FcγRIIIA. Methods for altering ADCC activity by antibody engineering have been described in the art, e.g., Shields RL et al., J Biol Chem. 2001. 276(9):6591-604; Idusogie EE et al., J Immunol. 2000. 164(8):4178-84; Steurer W et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE et al., J Immunol. 2001, 166(4):2571-5; Lazar GA et al., PNAS, 2006, 103(11):4005-4010; Ryan MC et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards JO, et al., Mol Cancer Ther. 2008, 7(8):2517-27; Shields RL et al., J. Biol. Chem, 2002, 277:26733-26740; Shinkawa T et al., J. Biol. Chem, 2003, 278:3466-3473.

[0225]

[0231] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein contain one or more amino acid substitutions that alter complement dependent cytotoxicity (CDC), for example, by improving or reducing C1q binding and / or CDC (see, e.g., WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351 for other examples of Fc region variants).

[0226]

[0232] In certain embodiments, the constant region of an antibody or antigen-binding fragment thereof provided herein comprises a substitution of one or more amino acid residues compared to SEQ ID NO:49 (i.e., the wild-type sequence) selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof. In certain embodiments, the constant region comprises the sequence of SEQ ID NO:51.

[0227]

[0233] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein contain one or more amino acid substitutions that improve pH-dependent binding to neonatal Fc receptor (FcRn). Such variants can have an extended pharmacokinetic half-life when bound to FcRn at acidic pH, allowing the variants to escape lysosomal degradation and then translocate and be released outside the cell. Methods for engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0228]

[0234] iii.Antigen-binding fragment

[0235] The anti-CLDN18.2 antibody conjugates provided herein can also include anti-CLDN18.2 antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-CLDN18.2 antibodies provided herein, including, for example, the exemplary antibodies whose CDR sequences are shown in Table 1 and their various variants (e.g., affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.).

[0229]

[0236] In certain embodiments, an anti-CLDN18.2 antigen-binding fragment provided herein is a diabody, a Fab, a Fab', a F(ab')2, an Fd, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody.

[0230]

[0237] A variety of techniques can be used to generate such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression by host cells such as E. coli (for example, for Fab, Fv, and ScFv antibody fragments), screening from a phage display library as discussed above (for example, for ScFv), and chemical coupling of two Fab'-SH fragments to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for generating antibody fragments will be apparent to those skilled in the art.

[0231]

[0238] In certain embodiments, the antigen-binding fragment is scFv. The generation of scFv is described, for example, in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. scFv can be fused to effector protein at either amino or carboxyl terminus to produce fusion protein (see, for example, Antibody Engineering, edited by Borrebaeck).

[0232]

[0239] In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment in the anti-CLDN18.2 antibody conjugate provided herein is bivalent, tetravalent, hexavalent, or multivalent. The term "valency" as used herein refers to the presence of a specified number of antigen-binding sites in a given molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" each refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. Any molecule that is more than bivalent is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0233]

[0240] A bivalent molecule can be monospecific when both binding sites are specific for binding to the same antigen or epitope. This allows for stronger binding to antigen or epitope than its monovalent counterpart in certain embodiments. Similarly, a multivalent molecule can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences, despite having the same specificity).

[0234]

[0241] Bivalent can also be bispecific, when two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific, when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0235]

[0242] bispecific antibody

[0243] In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein are bispecific. The term "bispecific" as used herein encompasses molecules with three or more specificities, and molecules with three or more specificities, i.e., multispecific. In certain embodiments, the bispecific antibodies and antigen-binding fragments provided herein can specifically bind to a first and a second epitope of CLDN18.2, or can specifically bind to CLDN18.2 and a second antigen. In certain embodiments, the first and second epitopes of CLDN18.2 are entirely different from each other or do not overlap. In certain embodiments, the bispecific antibodies and antigen-binding fragments thereof can simultaneously bind to both the first and second epitopes. In certain embodiments, the second antigen is different from CLDN18.2.

[0236]

[0244] In certain embodiments, the second antigen comprises a tumor antigen. As used herein, "tumor antigen" refers to tumor-specific antigens (e.g., antigens that are unique to tumor cells and not commonly found in non-tumor cells), tumor-associated antigens (e.g., found in both tumor and non-tumor cells, but differentially expressed in tumor cells), and tumor neo-antigens (e.g., expressed in cancer cells because somatic mutations change the protein sequence or create fusion proteins between two unrelated sequences).

[0237]

[0245] Preparation method

[0246] In another aspect, the disclosure provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising: 124 I, 123 I or 131 The present invention provides a method for preparing an anti-CLDN18.2 antibody conjugate provided herein, comprising reacting an iodide compound labeled with I in the presence of an enzymatic or chemical oxidizing agent.

[0238]

[0247] In some embodiments, the chemical oxidizing agent is N-bromosuccinimide, iodogen, or chloramine-T.

[0239]

[0248] In another aspect, the disclosure provides a method for producing a CLDN18.2 antibody, or antigen-binding fragment thereof, comprising the steps of conjugating the antibody or antibody conjugate with a chelator to obtain a chelator-antibody conjugate, and coupling the chelator-antibody conjugate with a metal radionuclide, e.g., 64 Cu or 89 and reacting the anti-CLDN18.2 antibody conjugate provided herein with Zr.

[0240]

[0249] In some embodiments, the chelating agent is DFO (delferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (NR-diethylenetriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-acetic acid), TRITA (1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid); TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid); and HETA (1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid), NETA ({ 4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), TACN-TM (N,N',N",tris(2-mercaptoethyl)1,4,7-triazacyclononane), TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]), CP256, PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid), porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and their derivatives.

[0241]

[0250] Pharmaceutical Compositions

[0251] The present disclosure further provides a pharmaceutical composition comprising anti-CLDN18.2 antibody conjugate and one or more pharma- ceutically acceptable carriers.In certain embodiments, the pharmaceutical composition provided herein is suitable for parenteral administration, for example, suitable for bolus injection, intravenous injection, or intratumoral injection.In certain embodiments, the pharmaceutical composition is in a unit dose injection form.

[0242]

[0252] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary agents, other ingredients known in the art, or various combinations thereof.

[0243]

[0253] Suitable ingredients can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorings, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, including one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments and conjugates provided herein reduces the oxidation of the antibodies or antigen-binding fragments. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided that include one or more antibody conjugates disclosed herein and one or more antioxidants, such as methionine.

[0244]

[0254] To further illustrate, pharma- ceutically acceptable carriers include, for example, aqueous vehicles such as Sodium Chloride Injection, Ringer's Solution, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics. The carrier may include drugs such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN®-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0245]

[0255] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition.The injectable pharmaceutical composition can be prepared in any conventional form, such as liquid solution, suspension, emulsion, or solid form suitable for producing liquid solution, suspension or emulsion.The preparation for injection can include sterile and / or non-pyrogenic solution ready for injection, sterile dry soluble, such as lyophilized powder, ready to be combined with solvent immediately before use, including subcutaneous tablets, sterile suspension ready for injection, sterile dry insoluble, ready to be combined with vehicle immediately before use, and sterile and / or non-pyrogenic emulsion.The solution can be either aqueous or non-aqueous.

[0246]

[0256] In certain embodiments, unit dose parenteral preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration are required to be sterile and non-pyrogenic, as is known and practiced in the art.

[0247]

[0257] In certain embodiments, a sterile, freeze-dried powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients to improve stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those of skill in the art, in one embodiment, at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of the anti-CLDN18.2 antibody conjugate or composition thereof. Overfilling the vial by a small amount (e.g., about 10%) beyond that needed for a dose or series of doses is permissible to facilitate accurate sampling and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0248]

[0258] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution, sterile water and / or non-pyrogenic water, or other liquid suitable carrier, is added to the lyophilized powder. The exact amount depends on the selected therapy being given, but may be empirically determined.

[0249]

[0259] Methods of Treating and Diagnosing CLDN18.2-Associated Conditions

[0260] The anti-CLDN18.2 antibody conjugate provided herein can be useful in the method for diagnosing or treating CLDN18.2-related condition.In certain embodiments, the CLDN18.2-related condition is cancer.In certain embodiments, the CLDN18.2-related condition is CLDN18.2-expressing cancer.

[0250]

[0261] In certain embodiments, the cancer is selected from gastric cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, and adenocarcinoma.

[0251]

[0262] Examples of cancer include, but are not limited to, non-small cell lung cancer (squamous / non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (e.g., basal, ductal, and lobular breast carcinoma), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, myeloma, mycosis fungoides, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, and other sarcomas. Myosarcoma, Lymphoid malignancies, Basal cell carcinoma, Adenocarcinoma, Sweat gland carcinoma, Medullary thyroid carcinoma, Papillary thyroid carcinoma, Pheochromocytoma, Sebaceous gland carcinoma, Papillary carcinoma, Papillary adenocarcinoma, Medullary carcinoma, Bronchogenic carcinoma, Hepatocellular carcinoma, Bile ductal carcinoma, Choriocarcinoma, Wilms' tumor, Cervical cancer, Testicular tumor, Seminoma, Classical Hodgkin's lymphoma (CHL), Primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Acute Lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumors, EBV-positive and -negative PTLD, and diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma , non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia and myelodysplasia, primary CNS lymphoma, spinal axis tumors, brain stem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinal germinomas.

[0252]

[0263] " CLDN18.2 expressing cancer " as used herein refers to any cancer or tumor with cancer cells expressing CLDN18.2. Examples of CLDN18.2 expressing cancer include gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), ovarian cancer, colon cancer, colorectal cancer, gastrointestinal mesenchymal tumor (GIST), gastrointestinal carcinoid tumor, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, appendix cancer; prostate cancer, kidney cancer (e.g. renal cell carcinoma), liver cancer, head and neck cancer, and gallbladder cancer and its metastasis, for example, gastric cancer metastasis such as Krukenberg tumor, peritoneal dissemination metastasis and lymph node metastasis.

[0253]

[0264] In certain embodiments, CLDN18.2 expressing cancer can be adenocarcinoma, for example, advanced adenocarcinoma.In certain embodiments, cancer is selected from gastric, esophageal, pancreatic duct, bile duct, lung and ovarian adenocarcinoma.In certain embodiments, CLDN18.2 expressing cancer includes gastric cancer, esophageal cancer, particularly lower esophageal cancer, esophageal-gastric junction cancer and gastroesophageal cancer.

[0254]

[0265] Methods for detecting, visualizing, diagnosing, staging, and monitoring CLDN18.2-associated conditions

[0266] The antibody conjugates provided herein are useful in detecting CLDN18.2 expression, particularly in vivo in a non-invasive manner.

[0255]

[0267] The antibody conjugate provided herein is also useful for visualizing, diagnosing, staging and monitoring CLDN18.2-related conditions.In certain embodiments, CLDN18.2-related conditions are cancer.In certain embodiments, CLDN18.2-related conditions are CLDN18.2-expressing cancer.

[0256]

[0268] The radionuclide conjugates of the antibodies provided herein can be used in vivo to provide information about (i) the expression of CLDN18.2, (ii) the distribution of CLDN18.2, and (iii) changes in the expression or distribution of CLDN18.2. Compared to currently available diagnostic methods for CLDN18.2, such as immunohistochemistry (IHC), the radionuclide conjugates of the antibodies provided herein have the advantages of non-invasive treatment, quantifiable, whole-body evaluation, and repeated dosing and evaluation at multiple time points.

[0257]

[0269] The anti-CLDN18.2 antibody conjugates provided herein can be used with in vivo nuclear imaging modalities to visualize target cells within the subject's body topography. In addition to diagnosing cancers associated with CLDN18.2 expression, the radionuclide conjugates of the anti-CLDN18.2 antibodies provided herein can also be used to stage and monitor the progression of cancer according to the methods provided herein.

[0258]

[0270] Suitable methods of in vivo nuclear imaging that may be used in accordance with the methods described herein include, but are not limited to, positron emission tomography (PET), combined PET / CT imagers, and single photon emission computed tomography (SPECT).

[0259]

[0271] In one aspect, the present disclosure provides a method of obtaining an image of a region of interest in a subject, the method comprising: a) administering to a subject an effective amount of an antibody conjugate provided herein and / or a pharmaceutical composition provided herein; b) subjecting the area of ​​interest of the subject to positron emission tomography (PET) or SPECT; c) identifying a detectable signal from the radionuclide in the subject; d) imaging the detectable signal, thereby obtaining an image of the site of interest in the subject; The present invention provides a method comprising:

[0260]

[0272] In some embodiments, the site of interest is a site expressing or suspected of expressing claudin18.2.

[0261]

[0273] In some embodiments, the site of interest is harboring or suspected of harboring a tumor, hi some embodiments, the site of interest may be the whole body, torso, head, or extremities.

[0262]

[0274] In another aspect, the disclosure provides a method of detecting or visualizing claudin18.2 expression in a subject in a non-invasive manner, comprising: a) administering to a subject an effective amount of an antibody conjugate provided herein and / or a pharmaceutical composition provided herein; b) subjecting the subject to positron emission tomography (PET) or SPECT; c) identifying a detectable signal from the radionuclide at a site of interest in the subject; d) determining or visualizing claudin18.2 expression in the site of interest of the subject based on the identified detectable signal; The present invention provides a method comprising:

[0263]

[0275] In some embodiments, a CLDN18.2 antibody conjugate provided herein may be injected intravenously into a subject. The subject may be scanned by PET at a certain time after injection.

[0264]

[0276] In certain embodiments, the method further comprises subjecting the subject to X-ray computed tomography (CT) to obtain a cross-sectional image of the body part under investigation. In certain embodiments, a combined CT and PET image can be obtained to show the expression and distribution of CLDN18.2 on the body's reference coordinate system.

[0265]

[0277] In some embodiments, the methods further comprise administering a therapeutically effective amount of an anti-claudin18.2 therapy to a subject identified as having claudin18.2 expression at the site of interest.

[0266]

[0278] The anti-claudin18.2 therapy may be any therapeutic agent that targets CLDN18.2, including, but not limited to, an anti-CLDN18.2 antibody, an anti-CLDN18.2 antibody drug conjugate, a small molecule compound that targets CLDN18.2, a nucleic acid that targets CLDN18.2, or a cell therapy that targets CLDN18.2.

[0267]

[0279] In some embodiments, the method further comprises determining a distribution of claudin18.2 expression in a site of interest in the subject based on the identified detectable signal.

[0268]

[0280] In some embodiments, the method further comprises determining the heterogeneity of claudin18.2 expression in the site of interest in the subject.

[0269]

[0281] In some embodiments, the site of interest is a tumor.

[0270]

[0282] In another aspect, the disclosure provides a method of monitoring therapeutic efficacy, responsiveness to treatment, or occurrence of resistance or recurrence, or metastasis in a subject who has been treated for a therapeutic period, in a non-invasive manner, comprising: a) administering to a subject an effective amount of an antibody conjugate provided herein and / or a pharmaceutical composition provided herein; b) subjecting the subject to positron emission tomography (PET) or SPECT; c) identifying a detectable signal from the radionuclide at a site of interest in the subject; d) determining post-treatment claudin18.2 expression at the site of interest in the subject based on the identified detectable signal; e) comparing the level or distribution of claudin18.2 expression after treatment to a baseline level or distribution of claudin18.2 expression, respectively, obtained from the subject prior to a treatment period to determine a post-treatment change in the level or distribution of claudin18.2 expression in the subject; f) determining therapeutic efficacy, responsiveness to treatment, or occurrence of resistance or recurrence, or metastasis based on the changes determined in step (e); The present invention provides a method comprising:

[0271]

[0283] In some embodiments, baseline claudin18.2 expression levels or distribution are determined using similar methods.

[0272]

[0284] In some embodiments, the baseline claudin18.2 expression level or distribution is determined prior to the therapeutic period (i.e., pre-treatment) by administering to a subject an effective amount of an antibody conjugate provided herein and / or a pharmaceutical composition provided herein, subjecting the subject to positron emission tomography (PET) or SPECT, identifying a detectable signal from a radionuclide at the site of interest in the subject, and determining baseline claudin18.2 expression at the site of interest in the subject based on the identified detectable signal.

[0273]

[0285] In some embodiments, elevated levels or spread of claudin18.2 expression indicates reduced therapeutic efficacy, reduced responsiveness to treatment, or the presence of resistance or recurrence. In some embodiments, metastasis can be indicated by the spread of claudin18.2 expression to sites where claudin18.2 expression was previously undetectable.

[0274]

[0286] In some embodiments, the subject has cancer.

[0275]

[0287] In some embodiments, the site of interest for determining baseline claudin18.2 expression is the same as or at least equivalent to the site of interest for determining post-treatment claudin18.2 expression. A site of interest is equivalent to another site of interest when both are of the same type of tissue or located within the same lesion.

[0276]

[0288] In some embodiments, the site of interest for the determination of baseline claudin18.2 expression may also be different from the site of interest for the determination of post-treatment claudin18.2 expression.

[0277]

[0289] Treatment Method

[0290] In another aspect, there is provided a method of treating a disease or condition in a subject that would benefit from modulation of CLDN18.2 activity, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a CLDN18.2 antibody conjugate and / or pharmaceutical composition provided herein.

[0278]

[0291] In certain embodiments, the disease or condition is CLDN18.2-related disease or condition.In some embodiments, the CLDN18.2-related disease or condition is cancer.In some embodiments, the CLDN18.2-related disease or condition is CLDN18.2-expressing cancer.

[0292] In certain embodiments, the CLDN18.2 antibody conjugate comprises a therapeutic radionuclide.

[0279]

[0293] In certain embodiments, the therapeutic radionuclide is 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P,33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb.

[0280]

[0294] In certain embodiments, the therapeutic radionuclide is 131 I, 177 Lu, 153 Sm, 223 Ra, 89 Sr, 90 Y, 213 Bi, 212 Pb, 166 Ho.

[0281]

[0295] The therapeutically effective amount of the CLDN18.2 antibody conjugates provided herein will depend on various factors known in the art, such as, for example, body weight, age, past medical history, current prescribed medications, the subject's health condition and the possibility of cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease development. The dosage may be proportionally increased or decreased by a skilled artisan (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements. The CLDN18.2 antibody conjugates disclosed herein may be administered by any route known in the art, for example, parenterally (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection).

[0282]

[0296] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a CLDN18.2-antibody conjugate provided herein.

[0283]

[0297] In certain embodiments, the subject is identified as having CLDN18.2-expressing cancer cells. The presence and / or expression level of CLDN18.2 on cancer cells can be determined by various methods known in the art, such as quantitative fluorescence cytometry, immunohistochemistry (IHC), qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, etc. The presence and / or expression level of CLDN18.2 in a subject can also be determined based on the methods provided herein.

[0284]

[0298] In certain embodiments, the subject has been diagnosed with a CLDN18.2-expressing cancer using the methods provided herein.

[0285]

[0299] In certain embodiments, the subject has been diagnosed with a CLDN18.2-expressing cancer using an antibody conjugate provided herein that includes a diagnostic radionuclide provided herein.

[0286]

[0300] In some embodiments, the CLDN18.2 antibody conjugate disclosed herein can be administered alone or in combination with one or more additional therapeutic means or agents.For example, the CLDN18.2 antibody conjugate disclosed herein can be administered in combination with a second therapeutic agent, such as a chemotherapeutic agent, an anticancer drug, radiation therapy, immunotherapy, an antiangiogenic agent, targeted therapy, cell therapy, gene therapy, hormone therapy, palliative care, surgery for the treatment of cancer (e.g., tumor removal), or one or more antiemetics or other treatments for complications resulting from chemotherapy.

[0287]

[0301] In certain of these embodiments, the CLDN18.2 antibody conjugates disclosed herein are administered in combination with one or more additional therapeutic agents, and in certain of these embodiments, the CLDN18.2 antibody conjugates and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, a CLDN18.2 antibody conjugate administered "in combination" with another therapeutic agent may not be administered simultaneously with the agent or in the same composition as the agent. A CLDN18.2 antibody conjugate administered before or after another agent is considered to be administered "in combination" with the agent, as the phrase "in combination" is used herein, even if the CLDN18.2 antibody conjugate and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with an antibody or antigen-binding fragment disclosed herein will be administered according to a schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, Vol. 57; Medical Economics Company; ISBN: 1563634457; Vol. 57 (November 2002)), or according to protocols well known in the art.

[0288]

[0302] kit

[0303] In another aspect, the present disclosure provides a kit comprising an anti-CLDN18.2 antibody conjugate provided herein or a composition provided herein.

[0289]

[0304] In some embodiments, the kits may be useful in detecting the presence or amount or distribution of CLDN18.2 in a subject, or may be useful in the diagnostic methods provided herein.

[0290]

[0305] In some embodiments, the kits may be useful in treating a CLDN18.2-expressing cancer in a subject or may be useful in the treatment methods provided herein.

[0291]

[0306] In some embodiments, the kit comprises a first composition comprising a first anti-CLDN18.2 antibody conjugate provided herein comprising a therapeutic radionuclide as disclosed herein, and a second composition comprising a second anti-CLDN18.2 antibody conjugate provided herein comprising a diagnostic radionuclide as disclosed herein. In some embodiments, the therapeutic radionuclide is 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb, and / or the diagnostic radionuclide is selected from the group consisting of: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 l, 124 l, 125 l, 131 l, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154’’1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 lr, 198 Au, 199 Au, 211At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac. In some embodiments, the first and second anti-CLDN18.2 antibodies are the same or different. In some embodiments, the first and second anti-CLDN18.2 antibodies both comprise a heavy chain variable region comprising HCDR1 comprising the sequence of SEQ ID NO: 1, HCDR2 comprising the sequence of SEQ ID NO: 19, and HCDR3 comprising the sequence of SEQ ID NO: 21, and a light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 14, LCDR2 comprising the sequence of SEQ ID NO: 16, and LCDR3 comprising the sequence of SEQ ID NO: 18.

[0292]

[0307] In some embodiments, the kits are for use in the methods provided herein.

[0293]

[0308] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent. The kit may be useful in treating, preventing, and / or ameliorating a CLDN18.2-associated disease.

[0294]

[0309] In another aspect, the disclosure provides the use of an anti-CLDN18.2 antibody conjugate provided herein, or a composition provided herein, in the manufacture of a pharmaceutical or diagnostic product for use in a method provided herein.

[0295]

[0310] In certain embodiments, the kit may further comprise a device capable of delivering the kit components through some other route, for example, a syringe for applications such as parenteral delivery, a syringe for injection of the composition into the subject's body. In certain embodiments, the anti-CLDN18.2 antibody conjugates provided herein may be provided in the form of a pre-filled syringe or autoinjector pen containing a sterile liquid formulation or lyophilized preparation of the antibody (e.g., Kivitz et al., Clin. Ther. 2006, 28:1619-29).

[0296]

[0311] The kit components may be packaged together or separated into two or more containers. In some embodiments, the container may be a vial containing a sterile lyophilized formulation of the composition suitable for reconstitution. The kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that may be used include, but are not limited to, pouches, trays, boxes, tubes, etc. The kit components may be packaged and maintained in a sterile condition within the container. Another component that may be included is instructions for use of the kit. EXAMPLES

[0297]

[0312] While the present disclosure has been particularly shown and described with reference to specific embodiments, some of which are preferred, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

[0298]

[0313] Example 1 Introduction

[0314] A series of studies have demonstrated that CLDN18.2, as a novel target for targeted therapy of gastric cancer, has a broad application prospect. How to maximize the potential beneficiaries with targeted and precise treatment, and how to rapidly and comprehensively evaluate the molecular pathological characteristics of CLDN18.2-positive gastric cancer patients are urgent problems to be solved.

[0299]

[0315] Solving this problem means putting forward higher requirements for CLDN18.2 detection technology, but there are still many limitations in the detection of CLDN18.2.

[0300]

[0316] (1) Single detection method. Currently, commercially available antibodies are only used for immunohistochemistry (IHC) detection of tissue paraffin sections and frozen tissue sections. In the context of insufficient discrimination ability and low sensitivity, they could not distinguish between CLDN18.2 or CLDN18.1 (another splice variant of Claudin18), because the amino acid sequences of CLDN18.2 and CLDN18.1 are highly homologous (91% of the amino acid sequences are identical).

[0301]

[0317] (2) The detection method is invasive. The single detection method results in CLDN18.2 detection samples being limited to tissue sections only, and the sampling is invasive and laborious, which has higher requirements for patients.

[0302]

[0318] (3) Heterogeneity of detection specimens: Heterogeneity of advanced gastric cancer is significant. We found that there may be heterogeneity in the tissue expression space and intensity of CLDN18.2, and there may be heterogeneity in the tissue and liquid biopsy specimens of CLDN18.2. Routine pathological molecular diagnostic specimens for advanced stage patients are mainly endoscopic biopsy specimens, which have low tissue content and limited sampling sites; therefore, a single endoscopic tissue specimen test may miss some potential beneficiaries due to the heterogeneity of CLDN18.2 expression.

[0303]

[0319] (4) Static test results: The study showed that there were significant differences in the intensity and spatial distribution of target expression and the immune microenvironment state of tumor tissue before and after drug treatment, which affected the efficacy of follow-up treatment. Due to limitations in sample collection and detection methods, it is difficult to support the exploration of dynamic monitoring for CLDN18.2. Therefore, the best solution for these problems is to explore and integrate non-invasive, comprehensive, real-time, and dynamic detection technologies for CLDN18.2 to make a comprehensive evaluation of the whole lesion, summarize the characteristics of potential beneficiaries combined with the therapeutic effect, and lead the diagnosis and treatment.

[0304]

[0320] Therefore, it is of great importance to develop a molecular probe with high specificity for the diagnosis of CLDN18.2 and the subsequent evaluation of immune checkpoint antibody response by using the comprehensive, non-invasive, real-time and dynamic characteristics of the latest molecular imaging technology. Although there are a large number of nuclide molecular probes focusing on HER2-positive gastric cancer and the technology is relatively mature, reports on CLDN18.2 nuclide probes are still blank at home and abroad. The present invention takes the CLDN18.2-targeting monoclonal antibody 18B10 as an example, and uses next-generation nuclide to label the 18B10 antibody. 124 I / 64 Cu / 89 Although Zr was used, in vivo studies showed that the antibody had significantly higher uptake in CLDN18.2-high expressing tumors, suggesting that the nuclide-labeled 18B10 antibody, as a specific probe targeting CLDN18.2, is a potential imaging agent for tumor immunotherapy screening, treatment process monitoring and efficacy evaluation.

[0305]

[0321] The present invention is, at least in part, based on the use of next-generation nuclides to label CLDN18.2-targeting monoclonal antibodies (taking 18B10 as an example). 124 I / 64 Cu / 89 Zr, as well as its preparation method and application.

[0306]

[0322] The concept of the present invention is as follows: 124 I(T 1 / 2 =4.2d), 64 Cu(T 1 / 2 =12.7h), 89 Zr(T 1 / 2 =78.4h) are novel solid-targeted PET nuclides, which have the prospects for broad clinical applications. 18 F(T 1 / 2 = 110 min) and 11 C(T 1 / 2 = 20.4 min) compared to 124 I / 64 Cu / 89 Zr has a relatively long half-life, which is conducive to long-term imaging studies and radionuclide transport, but low liver uptake, which is conducive to detection of liver lesions. Monoclonal antibodies that bind only one epitope (antigenic determinant) on an antigen molecule can be used as molecular probes to study the structure and function of antigens and to elucidate their mechanisms. It is easy to use nuclide-labeled monoclonal antibodies to determine the location and distribution of biopolymers (proteins, nucleic acids, enzymes, etc.) in vivo.

[0307]

[0323] To achieve the object of the present invention, in a first aspect, the present invention provides a next-generation nuclide-labeled CLDN18.2-targeting monoclonal antibody, which is a radionuclide-labeled monoclonal antibody. 124 I / 64 Cu / 89 Zr-labeled CLDN18.2-targeting monoclonal antibody.

[0308]

[0324] In a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: 124 I / 64 Cu / 89 A method for preparing a monoclonal antibody targeting Zr-CLDN18.2 (taking 18B10 as an example) is provided. By NBS method, 124I was labeled at the ortho-para position of the phenyl hydroxyl group in the molecular structure of the monoclonal antibody, and the method included the following: 0.5-1.5 mL of 0.1 M PB buffer at pH 7.2, 0.5-10 mg of 18B10, and 6-120 μg of N-bromosuccinimide were added in 25-90 KBq / μL of Na 124 0.5-1.0 mL of I solution was added and incubated at 37 °C for 60 s, and then 0.05-0.2 mL of 10% human serum albumin was added to stop the reaction; the final reaction solution was purified by PD-10 column to obtain the target product.

[0309]

[0325] 64 Cu / 89 Zr-labeled CLDN18.2-targeting monoclonal antibodies were obtained by using bifunctional chelating agents (taking 18B10 as an example): DFO equivalent to 6 moles of monoclonal antibody was added to 18B10 solution with a concentration of 2-5 mg / mL, and the pH value of the reaction system was adjusted to 8.5 with deionized 0.1 M sodium bicarbonate solution; DFO-18B10 was obtained by reacting at 4-37 °C for 4-24 h. 15 μg-2 mg of the labeled precursor was added to 0.1-1.0 mL of 0.1 M sodium acetate solution with pH 5.5, and then 25-100 MBq of fresh 18B10 was added to 18B10 solution with a concentration of 2-5 mg / mL. 64 Cu / 89 Zr solution was added and the pH value was adjusted to 7.0; the temperature was controlled at 40° C. for 30 min; the final reaction solution was purified by PD-10 column to obtain the target product.

[0310]

[0326] When the labeling rate was over 95% and purified by PD-10 column, the radiochemical purity of the target product was 98.76%.

[0311]

[0327] Before use, the PD-10 column was first equilibrated with 0.01M PBS solution at pH 7.4, 5mL of PBS was added each time, and the column was dried at gravity flow rate, which was repeated 5 times; then the target product was purified by 0.01M PBS solution at pH 7.4.

[0312]

[0328] 124In vitro stability analysis of I-18B10 124 It was shown that I-18B10 could maintain good stability in 0.01M PBS solution at pH 7.4 at 25°C for 48 hours, and the radiochemical purity was 97.89±0.05%.

[0313]

[0329] In a third aspect, the present invention relates to a method for the preparation of tumor imaging and therapeutic agents targeting CLDN18.2. 124 I / 64 Cu / 89 The application of Zr-CLDN18.2 targeting monoclonal antibody (taking 18B10 as an example) is provided.

[0314]

[0330] In a fourth aspect, the present invention relates to a method for preparing a PET / CT molecular imaging probe. 124 I / 64 Cu / 89 The application of Zr-CLDN18.2 targeting monoclonal antibody (taking 18B10 as an example) is provided.

[0315]

[0331] In a fifth aspect, the present invention provides a CLDN18.2-targeted tumor imaging agent comprising a nuclide-labeled CLDN18.2-targeting monoclonal antibody.

[0316]

[0332] New generation of nuclide-labeled monoclonal antibodies ( 124 I-18B10) can specifically bind to CLDN18.2 on the tumor cell surface; in normal mice, there was no specific distribution in organs and tissues except blood; in PET imaging studies of PDX-bearing mice, as shown in Figure 4, both CLDN18.2-positive and CLDN18.2-negative tumor-bearing models showed 18 F-FDG uptake was extremely low in mice bearing CLDN18.2-positive PDXs. 124The distribution of I-18B10 changed dynamically with time: the uptake signal in the tumor site was significantly elevated at 60 h compared to 2 h; at 120 h, the uptake signal in the cardiac blood pool disappeared, and only the signal in the tumor could be observed. 124 in mice bearing I-18B10 and CLDN18.2 positive PDXs 124 There was no specific accumulation of I-IgG. 124 The I-18B10 probe was specifically accumulated in CLDN18.2-positive tumors, and was shown to have the characteristics of high sensitivity and high specificity. 124 The I-18B10 probe may in the future be used as an imaging agent to detect the expression of CLDN18.2 in patient tumors in a non-invasive manner.

[0317]

[0333] In summary, the present invention has at least the following advantages and beneficial effects: The novel PET molecular probe provided by the present invention 124 I / 64 Cu / 89 The Zr-CLDN18.2-targeting monoclonal antibody has stable characteristics, good imaging effect, high affinity to CLDN18.2 and high functional activity, and is expected to be used for real-time and non-invasive monitoring of CLDN18.2 expression in systemic lesions, monitoring the heterogeneity of CLDN18.2 expression in the same and different lesions, and observing the changes of CLDN18.2 expression during treatment, thereby screening patients, monitoring therapeutic effects, providing early warning of drug resistance and / or recurrence or metastasis, and realizing personalized and precise treatment of targeted tumor drugs.

[0318]

[0334] In the present invention, next-generation PET nuclides 124 I / 64 Cu / 89Zr is used, which can realize the labeling of monoclonal antibodies and has little effect on the activity of monoclonal antibodies; in addition, the non-specific uptake in the liver is low, which is conducive to the detection of liver lesions; and the whole-body background is low, which has a good tumor to non-tumor ratio and is conducive to the observation of tumor lesions.

[0319]

[0335] Example 2 Preparation of next-generation nuclide-labeled monoclonal antibodies targeting CLDN18.2 ( 124 Take I-18B10 as an example)

[0336] The CLDN18.2-targeting monoclonal antibody 18B10 used in this study was supplied by MabSpace Biosciences (Suzhou) Co., Ltd., which is cross-reactive with both human and mouse CLDN18.2 and can be tested in mice for preclinical studies. PD-10 preloaded gel columns were purchased from GE (USA).

[0320]

[0337] 124 I-18B10 was prepared by NBS reaction: 0.8 mL of 0.1 M PB buffer at pH 7.2, 0.1 mL of 18B10 monoclonal antibody solution (prepared in HO) (29.2 mg / mL) and 36 μg of NBS were added to 59.2 KBq / μL of Na 124 The mixture was added in turn to 1.0 mL of I solution, incubated at 37° C. for 60 seconds, and then 0.1 mL of 10% human serum albumin was added to stop the reaction; the final reaction solution was purified by PD-10 column to obtain the target product.

[0321]

[0338] Before use, the PD-10 column was first equilibrated with 0.01M PBS solution at pH 7.4, 5mL of PBS was added each time, and the column was dried at gravity flow rate, which was repeated 5 times; then the target product was purified by 0.01M PBS solution at pH 7.4.

[0322]

[0339] Radio-TLC and Radio-HPLC were used to determine the radiolabeling rate and radiochemical purity. Radio-TLC detection: Free Na with radioactivity of 37-74 kBq (1-2 μCi). 124 I 2 μL and purified 124 I-18B10 was added to 20μL of saturated EDTA. 2μL of the above solution was dropped 1cm from the bottom of Xinhua No.1 filter paper and placed in a saline development system. After completion of development, the filter paper was taken out and dried for Radio-TLC detection. Radio-HPLC detection: Free Na with radioactivity of 37-74kBq (1-2μCi) was detected. 124 I 2 μL and purified 124 I-18B10 was diluted in 50 μL of 0.01 M PBS pH 7.4 for Radio-HPLC analysis. Analytical conditions: Agilent Bio SEC-3 gel filtration / volume exclusion chromatography column, flow rate 1 mL / min, mobile phase 0.01 M PBS pH 7.4.

[0323]

[0340] target product 124 The radiolabeling rate and radiochemical purity of I-18B10 were 97.94% and 98.76%, respectively. 124 The radiochemical purity of I-18B10 remained approximately 98% after 48 hours in PBS (Figure 1).

[0324]

[0341] Example 3: Incorporation of next-generation nuclide-labeled monoclonal antibodies targeting CLDN18.2 ( 124 Take I-18B10 as an example)

[0342] Through transfection of CLDN18.2 into MKN45 cells and screening, we obtained the MKN45-CLDN18.2 high cell line with high expression of CLDN18.2. MKN45 cells were used as negative control cells. The seeded cells were uniformly cultured overnight in a 24-well plate to allow the cells to fully adhere to the wall. On the second day, 1 mCi of 124 I-18B10 or 124I-hIgG was added and incubated for 10, 30, 60, and 120 min, respectively. In the blocking group, cold precursor 18B10 was added at 0.5 mg / well to block the uptake signal. After incubation, the cells were washed three times with PBS and the lysates were collected. The uptake signal was detected and analyzed by a γ counter.

[0325]

[0343] The results (Figure 2) showed that MKN45-CLDN18.2 cells were 124 The uptake rate of I-18B10 was high and increased over time. When cold precursors were added for blocking, the uptake rate was significantly decreased, indicating that the uptake signal was specific for CLDN18.2. 124 The uptake of I-hIgG by MKN45 cells was also 124 The uptake of I-18B10 was also low in both cases.

[0326]

[0344] Example 4 Next-generation nuclide-labeled monoclonal antibody targeting CLDN18.2 ( 124 Biodistribution of I-18B10 in normal mice (taking I-18B10 as an example)

[0345] 124 I-18B10 (0.74MBq, 200μL) was injected into normal Kunming mice via tail vein. The mouse heart, liver, lung, kidney, spleen, stomach, bone, muscle, large intestine, small intestine, brain, blood, and other organs and tissues were isolated 2 hours, 24 hours, 60 hours, and 120 hours after injection. After grinding, the uptake signal was detected and analyzed by a gamma counter. 124 The ID% / g of the I-18B10 probe was obtained.

[0327]

[0346] In normal mice (Figure 3), blood uptake was highest at all time points. In addition to blood, uptake in liver, kidney, lung and spleen at 2 hours was higher than uptake in other organs and tissues. Uptake in all organs decreased over time. Compared to blood, uptake in other organs tended to be lower.

[0328]

[0347] Example 5 Next-generation nuclide-labeled monoclonal antibodies targeting CLDN18.2 in PDX tumor-bearing mice ( 124 PET imaging of I-18B10 as an example

[0348] CLDN18.2 positive PDXs (positive intensity 3+) and CLDN18.2 negative PDXs were selected for inoculation by immunohistochemistry (IHC). The whole tumor tissue was subcutaneously injected into the right lower leg of nude mice. When the diameter of the PDX tumor was 0.8 cm, the tumor-bearing mice were divided into several groups. The CLDN18.2 positive PDX-bearing mice were divided into three groups: 18 F-FDG group, 124 I-18B10 group and 124 These three groups were then administered the following via the tail vein: 18 F-FDG, 18.5 MBq 124 I-18B10, and 18.5MBq 124 I-hIgG was injected into mice. CLDN18.2-negative PDX tumor-bearing mice were also divided into three groups. At 2, 60, and 120 hours after injection, animals were anesthetized with isoflurane (2% isoflurane-30% oxygen / air) and imaging was performed with microPET / CT.

[0329]

[0349] As shown in Figure 4, 18 F-FDG uptake was extremely low in both CLDN18.2-positive and -negative tumor-bearing models. 124 The distribution of I-18B10 changed dynamically with time: at 2 h, uptake signals were detected in the cardiac blood pool and CLDN18.2-positive tumor sites; at 60 h, uptake signals in the cardiac blood pool were significantly decreased, while those in the tumors were increased; at 120 h, uptake signals in the cardiac blood pool disappeared, and only signals in the tumors could be observed. In CLDN18.2-negative PDX-bearing mice, uptake signals in all tissues and tumors were significantly increased. 124The uptake signals of I-18B10 were quite similar at different time points, indicating that there was no specific accumulation in CLDN18.2-negative tumors. As in the negative control, in CLDN18.2-positive PDX-bearing mice 124 The uptake signal of I-IgG was found to be higher in the cardiac blood pool than in the tumor at each time point. 124 The results show that the I-18B10 probe specifically accumulated in CLDN18.2-positive tumors and still had significant signals after long-term (120 h) imaging. This probe has the characteristics of high sensitivity and specificity, and can be used as an imaging agent to non-invasively detect the expression of CLDN18.2 in patients' tumors in the future.

[0330]

[0350] Next-generation nuclides provided by the present invention 124 I / 64 Cu / 89 The Zr-labeled CLDN18.2 targeting monoclonal antibody has stable characteristics, good imaging effect, high affinity to CLDN18.2 and high functional activity, which is conducive to the diagnosis and accurate staging of CLDN18.2 positive tumor lesions. Preclinical animal studies confirm that the monoclonal antibody of the present invention has the potential to be a CLDN18.2 targeting imaging agent with good application prospects.

[0331] Example 6: Epitope mapping of selected antibodies via site-directed mutagenesis at amino acids in CLDN18.2 that differ from CLDN18.1

[0351] 1. Generation of Human CLDN18.2-mRFP and Human CLDN18.1-mRFP Constructs

[0352] cDNAs encoding human CLDN18.1 (amino acids 1-261, SEQ ID NO: 31)-mRFP1 (amino acids 1-225) and human CLDN18.2 (amino acids 1-261, SEQ ID NO: 30)-mRFP1 (amino acids 1-225) were synthesized in vitro (SEQ ID NO: 52 and SEQ ID NO: 53 are their amino acid sequences, respectively). The PCR products were then cloned into the pcDNA3.1(+) vector by the method of homologous recombination using Syno Assembly Mix Reagent (Synbio) according to the manufacturer's instructions. The plasmids were purified by using QIAGEN Plasmid Mega Kit (QIAGEN).

[0332]

[0353] According to the sequences of human CLDN18.1 and CLDN18.2 (Genbank accession numbers: splice variant 1 (CLDN18.1): NP_057453, NM_016369, and splice variant 2 (CLDN18.2): NM_001002026, NP_001002026), eight different amino acids are located between 28 and 70, which may be determinants of specific binding to human CLDN18.2 but not to CLDN18.1. Using the wild-type human CLDN18.2-mRFP plasmid generated above as a template, two segments of the integrated sequence were generated using primers. The variants of human CLDN18.2-mRFP, in which a single amino acid was changed to that of human CLDN18.1 at the indicated positions, were amplified by overlapping PCR using primers. The specific mutations are on Q29M, N37D, A42S, N45Q, Q47E, E56Q, G65P, and L69I. Variants of human CLDN18.1-mRFP with single amino acid changes at the indicated positions were amplified by overlap PCR using primers. The specific mutations are on M29Q, D37N, S42A, Q45N, E47Q, Q56E, P65G, I69L. The PCR products were then cloned into the pcDNA3.1(+) vector by the method of homologous recombination. The human CLDN18.2-mRFP variants were identified and confirmed by sequencing individual positive clones.

[0333]

[0354] These plasmids of mutant and wild-type human CLDN18.2-mRFP or human CLDN18.1-mRFP were then transfected into HEK293 cell lines. 6 HEK293 cells were seeded at 60%-80% ratio in a 60 mm dish for transfection. 10 μg of DNA in 400 μl of 1× HBS and 10 μl of 25 kDa linear PEI transfection reagent (dissolved in 1× HBS, 1 mg / ml stock solution) were mixed to reach a DNA / PEI ratio of 1:2.5. The mixture was then added dropwise to the HEK293 cell culture. After 6-8 hours, the transfected cells were replaced with complete DMEM overnight. 24 hours after transfection, cells were harvested for FACS analysis using chimeric antibodies.

[0334]

[0355] Amino acid sequence of human CLDN18.1 (SEQ ID NO:31) MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0356] Amino acid sequence of human CLDN18.1-mRFP1 (SEQ ID NO:52): MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTS GIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDG GARTEDEVQSYPSKHDYVMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA

[0357] Amino acid sequence of human CLDN18.2 (SEQ ID NO:30) MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0358] Amino acid sequence of human CLDN18.2-mRFP1 (SEQ ID NO:53): MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTS GIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDG GARTEDEVQSYPSKHDYVMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA

[0359] 2. Binding of CLDN18.2 Chimeric Antibody to Site-Mutated HEK293-CLDN18.2 or HEK293-CLDN18.1 Cells

[0360] Transfected HEK293-CLDN18.2 or HEK293-CLDN18.1 cells were resuspended at a density of 10^5 cells / well, 100 μl / well in PBS containing 2% BSA. Cells were washed three times with FACS wash buffer (PBS + 2% FBS) and incubated with 10 μg / ml of chimeric antibody per well and 100 μl / well of IMAB362 for 30 min at 4°C. Next, cells were washed three times with FACS wash buffer and incubated with 100 μl / well of goat anti-hIgG(H+L)-FITC (1:200 dilution) for another 30 min at 4°C. Finally, cells were washed three times with FACS wash buffer and analyzed by flow cytometry. RFP-positive cells were used for control gating to analyze binding to CLDN18.2-transfected cells.

[0335]

[0361] IMAB362 was expressed according to the sequence disclosed in US2009169547A1. Single cell clones with the highest signal were selected and amplified for cell banking.

[0336]

[0362] Heavy chain variable region of IMAB362 (SEQ ID NO:72) QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS

[0363] Light chain variable region of IMAB362 (SEQ ID NO:73)

[0364] DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK

[0365] The percentage of binding signals of these chimeric antibodies to mutant CLDN18.2 variants compared to the percentage of binding signals of wild type was calculated and summarized in Table 4. As shown in Figures 5A-5B, the binding of 18B10-C (i.e., chimeric antibody of 18B10 fusion fused to human IgG1 constant region) was completely lost when E56 was mutated to Q. This change was also true for other chimeric antibodies except IMAB362 and 59A9-C. In addition, we found that other amino acids such as A42, N45 also contributed to some extent to the binding of IMAB362 and other antibodies, but not 18B10-C.

[0337]

[0366]

[0338] [Table 5]

[0339] Example 7: Generation and characterization of humanized antibodies

[0367] 1. Generation, Expression, and Purification of Humanized Antibodies

[0368] 18B10

[0369] Human germline framework sequences VK / 4-1 for the light chain and VH / 1-46 for the heavy chain, respectively, were used for CDR grafting.

[0340]

[0370] Heavy chain (HC) variants 1, 2 and 3 were obtained by directly grafting the three CDRs onto the germline sequence (18B10 HC germline, SEQ ID NO: 23) and the R71I, T73K back mutations (Hu18B10_Ha, SEQ ID NO: 25) for HC variant 1, onto the above germline sequence and the R71I, T73K, T28S, M69L back mutations (Hu18B10_Hb, SEQ ID NO: 27) for HC variant 2, and onto the above germline sequence and the R71I, T73K, T28S, M69L, R38K, M48I back mutations (Hu18B10_Hc, SEQ ID NO: 29) for HC variant 3, respectively.

[0341]

[0371] (1) 18B10 HC germline sequence:

[0372] VH / 1-46 (18B10-germline, SEQ ID NO:23): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR

[0373] VH / 1-46 variant 1 (Hu18B10_Ha, SEQ ID NO:25): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTSYNQKFKGRVTMTIDKSTSTVYMELSSLRSEDTAVYYCARMYHGNAFDYWGQGTTVTVSS

[0374] VH / 1-46 variant 2 (Hu18B10_Hb, SEQ ID NO:27): QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTSYNQKFKGRVTLTIDKSTSTVYMELSSLRSEDTAVYYCARMYHGNAFDYWGQGTTVTVSS

[0375] VH / 1-46 variant 3 (Hu18B10_Hc, SEQ ID NO:29): QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYNMNWVKQAPGQGLEWIGNIDPYYGGTSYNQKFKGRVTLTIDKSTSTVYMELSSLRSEDTAVYYCARMYHGNAFDYWGQGTTVTVSS

[0376] Light chain (LC) variants 1 and 2 were obtained by directly grafting the three CDRs onto the germline sequence (18B10 LC germline, sequence number 24), with no back mutations for variant 1 (Hu18B10_La, sequence number 26) and S63T, I21M for LC variant 2 (Hu18B10_Lb, sequence number 28), respectively.

[0342]

[0377] (2) Germline sequence of 18B10 LC:

[0378] VK / 4-1 (18B10 LC germline, SEQ ID NO:24) DIVMTQSPDSLAVSLGERATINCKSSQNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP

[0379] VK / 4-1 variant 1 (Hu18B10_La, SEQ ID NO:26) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNLKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGGGTKVEIK

[0380] VK / 4-1 variant 2 (Hu18B10_Lb, SEQ ID NO:28) DIVMTQSPDSLAVSLGERATMNCKSSQSLLNSGNLKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGGGTKVEIK

[0381] Combinations of the above heavy and light chain variable regions generate the following humanized 18B10 antibodies: 18B10-HaLa (having a VH of SEQ ID NO:25 and a VL of SEQ ID NO:26), 18B10-HbLa (having a VH of SEQ ID NO:27 and a VL of SEQ ID NO:26), 18B10-HcLa (having a VH of SEQ ID NO:29 and a VL of SEQ ID NO:26), 18B10-HaLb (having a VH of SEQ ID NO:25 and a VL of SEQ ID NO:28), 18B10-HbLb (having a VH of SEQ ID NO:27 and a VL of SEQ ID NO:28), 18B10-HcLb (having a VH of SEQ ID NO:29 and a VL of SEQ ID NO:28).

[0343]

[0382] The humanized variants of the 18B10 heavy and light chains are linked to the human IgG1 heavy chain constant region and the kappa light chain constant region as shown below:

[0383] Human IgG1 heavy chain constant region (SEQ ID NO:49): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0384] Human kappa light chain constant region (SEQ ID NO:50): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0385] The variable regions of the heavy and light chain cDNAs mentioned above were synthesized and fused with the constant regions of human IgG1 and human kappa. The heavy and light chains of the selected antibody genes were cloned into expression vectors, and large-scale DNA was prepared using the Plasmid Maxiprep System from Qiagen. Transfection was performed using ExpiFectamine™ CHO Reagent from Invitrogen according to the manufacturer's protocol. The supernatant was harvested when the cell viability was approximately 60%. The cell culture supernatant was filtered through a 0.22 μm filtration capsule to remove cell debris. The supernatant was loaded onto a pre-equilibrated Protein A affinity column. The Protein A resin inside the column was then washed with equilibration buffer (PBS), and the antibody was eluted using 25 mM citrate (pH 3.5). The pH was adjusted to approximately 6.0-7.0 using 1 M Tris base (pH 9.0). Endotoxin was controlled to less than 1 EU / mg. The purified antibodies were then characterized by SDS-PAGE and SEC-HPLC.

[0344]

[0386] 2. Binding to human and mouse CLDN18.2

[0387] Log-phase HEK293-CLDN18.2 and NUGC4 cells were resuspended in PBS. After washing the cells 3x by using FACS wash buffer (PBS + 2% FBS), 100 μl / well of diluted humanized antibodies ranging from 400 nM to 0.002 nM were added to each well for 30 min incubation at 4°C. Again, the cells were washed 3 times by using FACS wash buffer and then incubated with 100 μl / well of goat anti-mIgG-FITC (1:400 dilution) for another 30 min at 4°C. After a final 3x wash with FACS wash buffer, the cells were analyzed by flow cytometry.

[0345]

[0388] To screen the best one, all humanized antibody variants were tested directly against chimeric antibody. All variants retained their binding completely. Then, 18B10-HaLa, which had only one back mutation, was tested for its binding to HEK293 mouse CLDN18.2 cells. 18B10-HaLa could bind well to mouse CLDN18.2 with better potency and higher MFI than IMAB362, indicating that 18B10-HaLa had good cross-reactivity to mouse.

[0346]

[0389] 3.Affinity analysis of humanized CLDN18.2 antibody by KinExA

[0390] 18B10-HaLa and IMAB362 were evaluated in a head-to-head comparison by KinExA for their affinity binding to CLDN18.2 expressing cells. Following the instructions of KinExA 4000 (Sapidyne Instruments Inc.), 200mg of PMMA hard beads (Sapidyne, #440176) were coated with 30μg of goat anti-human IgGFc antibody for 2h, then blocked with BSA 10mg / ml for 1h. Two gastric cell lines, NUGC4 and KATOIII (ATCC, Cat#HTB-103), were harvested in log phase and mixed with 0.2nM 18B10-HaLa or IMAB362. The cell-antibody mixture was diluted 2-fold using 0.2nM of 18B10-HaLa or IMAB362 and incubated at room temperature for 3h. The amount of free antibody increased with dilution. The free antibodies were captured by goat anti-human IgGFc coated beads and subsequently labeled with Alexa Fluor 647 anti-human IgG 1 μg / ml for readout.

[0347]

[0391] The binding affinity of each antibody is summarized in Table 5. The Kd of 18B10-HaLa binding to NUGC4 cells and KATOIII was approximately 0.3 nM, which was more than 8-fold higher than the Kd of IMAB362, consistent with the FACS binding results above.

[0348]

[0392]

[0349] [Table 6]

[0350]

[0393] 4. CDC Assay on HEK293-CLDN18.2 Cells

[0394] 18B10-HaLa was tested in head-to-head comparison with IMAB362 in a CDC activity assay. 18B10-HaLa had more than 20-fold higher CDC activity than IMAB362. The percentage of 18B10-HaLa-dependent specific cell killing reached 86% at a concentration of 0.3 μg / ml, while IMAB362 had no cell killing at the same concentration.

[0351]

[0395] 5. Binding and cytotoxic effects on MKN45-CLDN18.2 cells

[0396] The cell binding assay was the same as above. All humanized variants of 18B10 bound to cells with similar affinity as chimeric 18B10. 18B10-HaLa, which has only one back mutation, was selected for further ADCC activity studies.

[0352]

[0397] ADCC activity was tested using Jurkat-NFAT-luc-FcγRIIIA-V176 cells as effector cells and MKN45-CLDN18.2 cells as target cells. 18B10-HaLa had a much lower EC50 (0.05 μg / ml) than IMAB362, which was consistent with the EC50 of chimeric 18B10.

[0353]

[0398] 6. Binding and cytotoxic effects on NUGC4 cells

[0399] Cell binding and ADCC assays were the same as above. 18B10-HaLa showed much better ADCC potency (EC50 approximately 0.59 μg / ml) compared to IMAB362.

[0354]

[0400] 7. ADCC assay using NUGC4 as target cells and PBMC as effector cells

[0401] Log-phase NUGC4 cells were resuspended in RPMI1640 containing 10% FBS. Cells were pre-seeded at 1x10^4 cells per well in 96-well U-bottom plates. Anti-CLDN18.2 antibody and IMAB362 were gradient-diluted in RPMI1640 containing 10% FBS and added to the above plates at final concentrations from 200 to 0.2μg / ml and incubated at 37℃ for 30 minutes. Frozen PBMCs from Miao Shun(Shanghai)Biological & Technology Co.,Ltd were removed from liquid nitrogen and immediately placed in a 37℃ water bath. After centrifugation, cells were resuspended in RPMI1640+10%FBS and then seeded at 40x10^4 cells per well in the above 96-well U-bottom plates. The plates were then placed in an incubator at 37℃ for 5 hours.

[0355]

[0402] After incubation, the plates were equilibrated to 22°C. LDH was detected by using the Promega CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (G7892) or the CytoTox 96® Non-Radioactive Cytotoxicity Assay (G1780). After adding Lysis, Reagent and Stop Solutions according to the manufacturer's instructions, fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm (G7892) or absorbance was measured at 490 nm or 492 nm (G1780).

[0356]

[0403] 18B10-HaLa showed much better ADCC potency than IMAB362. The EC50 may not be calculated accurately due to lack of fit to the regression curve.

[0357]

[0404] 8. Epitope mapping of selected antibodies using site-directed mutagenesis in human CLDN18.2

[0405] Using the same method and human CLDN18.2-mRFP plasmid, 42 amino acids between 28 and 80 of human CLDN18.2 listed below were replaced one at a time with alanine. These variants were amplified by overlap PCR using primers: Specific mutations are: Q28A, Q29A, W30A, S31A, T32A, Q33A, D34A, L35A, Y36A, N37A, N38A, V40A, T41A, V43A, F44A, N45A, Y46A, Q47A, L49A, W50A, R51A, S52A, V54A, R55A, E56A, E56A, S57A, S58A, F60A, T61A, E62A, R64A, Y66A, F67A, T68A, L69A, L70A, L72A, M75A, L76A, Q77A, V79A, R80A. The PCR product was then cloned into the pcDNA3.1(+) vector by the method of homologous recombination using Syno Assembly Mix Reagent (Synbio) according to the manufacturer's instructions. The plasmid was purified by using QIAGEN Plasmid Mega Kit (QIAGEN).

[0358]

[0406] These mutant and wild-type CLDN18.2-mRFP plasmids were then transfected into HEK293 cells, and cells were analyzed by flow cytometry 24 hours after transfection.

[0359]

[0407] When W30, L49, W50, and E56 were mutated to A, the binding of 18B10-HaLa was completely lost (binding percentage <10%), indicating that these four amino acids are crucial for its binding to human CLDN18.2. Among them, E56 is the most important one constituting the binding epitope. Besides these four crucial amino acids, several other amino acids, such as R51, F60, E62, and R80, also affect the binding (binding percentage between 10% and 25%) when replaced with alanine. The binding of 59A9-C to the site-mutated CLDN18.2 was only partially dependent on E56 (binding percentage about 22%). The binding percentage of the mutant CLDN18.2 to the antibody compared to the wild type is summarized in Table 6.

[0360]

[0408]

[0361] [Table 7-1]

[0362] [Table 7-2]

[0363]

[0409] Example 8 Next-generation nuclide-labeled monoclonal antibody targeting CLDN18.2 ( 124 Biodistribution of I-18B10 in PDX mice (taken as an example)

[0410] 124 I-18B10 (0.74MBq, 200μL per group) was injected via the tail vein into patient-derived xenograft (PDX) models (confirmed by claudin18.2 overexpression, provided by the Department of Digestive Oncology, Peking University Cancer Hospital) formed by transplanting tumor tissue from a gastric cancer patient's tumor into NSG mice. Biodistribution experiments were performed in PDX mice for the test groups. 124 I-18B10 and the blocking group 124The mice were divided into two groups, including those co-administered with I-18B10 and unlabeled 18B10 (200 mg / mouse). The mouse heart, liver, lung, kidney, spleen, stomach, bone, muscle, large intestine, small intestine, brain, and tumor were isolated 240 hours after injection. After grinding, the uptake signal was detected and analyzed by a γ counter. 124 The ID% / g of the I-18B10 probe was obtained.

[0364]

[0411] In PDX mice (Figure 6), 124 The uptake of I-18B10 was the highest compared to the other groups. Due to competition with unlabeled 18B10, the blocking group showed a significant decrease in uptake in both normal and tumor tissues. The results show that: 124 The results showed that I-18B10 was concentrated in tumor cells, which is a useful tool for disease diagnosis. 124 Supports the use of I-18B10.

[0365]

[0412] Example 9 Next-generation nuclide-labeled monoclonal antibodies targeting CLDN18.2 in patients ( 124 Mapping of I-18B10 as an example

[0413] In an exploratory clinical study, patients diagnosed with claudin18.2-expressing cancer were 124 Within 1 week before or after I-18B10 PET / CT imaging 124 I-18B10 PET / CT scan and 18 F-FDG PET / CT was performed (11.1–18.5 MBq per patient, by intravenous injection). 124 For I-18B10 PET imaging, 124 The patient's thyroid was shut off by taking Lugol's potassium iodide (10 drops each time, three times a day) 3 days before and 7 days after administration of I-18B10. 124 I-18B10 PET / CT scan, 124Following I-18B10 administration, PET images were acquired using a Siemens Biograph mCT Flow 64 scanner (Erlangen, Germany) at 2, 24, 72, and 96 hours. PET images were acquired from head to foot or upper thigh at scan speeds of 0.8 mm / s, 0.5 mm / s, 0.4 mm / s, 0.3 mm / s, and 0.2 mm / s at 2, 24, 48, and 72 hours, respectively, and reconstructed using expectation maximization with ordered subsetting.

[0366]

[0414] In patients 124 The biodistribution of I-18B10 was derived from the images of the first five patients (Figure 7). The thyroid gland was well blocked in the patients. The tracer was concentrated in the blood after injection and decreased over time, while the concentration in the spleen and liver increased over time. The spleen was the organ with the highest activity at 24, 48, and 72 hours. There was also tracer uptake in the kidneys, with lower uptake in the brain, lungs, and bones. No obvious tracer uptake was seen in the normal stomach wall. In addition, Figure 8 shows that 124 I-18B10 accumulated in the patient's ovarian lesion, indicating that this was claudin18.2-expressing cancer that had metastasized to the ovary. 18 Compared with the results of F-FDG PET / CT, 124 I-18B10 PET / CT 24 hours and 124 I-18B10 PET / CT 48-hour results: 124 I-18B10 had higher uptake in claudin18.2-expressing cancer tissues in ovarian lesions ( 18 F-FDG PET / CT SUVmax: 3 (left), 2.9 (right); 124 I-18B10 PET / CT 24-hour SUVmax: 25.1 (left), 22.2 (right); 124 I-18B10 PET / CT showed 48-hour SUVmax: 12.5 (left), 17.6 (right).

Claims

1. An anti-CLDN18.2 antibody conjugate comprising an anti-CLDN18.2 antibody or an antigen-binding fragment thereof conjugated to a radionuclide, including a therapeutic radionuclide or a diagnostic radionuclide.

2. The therapeutic radionuclide is １１１ In, １１１ｍ In, １７７ Lu, ２１２ Bi, ２１３ Bi, ２１１ At, ６２ Cu, ６４ Cu, ６７ Cu, ９０ Y. １２５ I, １３１ I, ３２ P. ３３ P. ４７ Sc, １１１ Ag, ６７ G.A. １４２ P.R. １５３ Sm, １６１ Tb, １６６ Dy, １６６ Ho, １８６ Re, １８８ Re, １８９ Re, ２１２ Pb, ２２３ Ra, ２２５ A c, ５９ Fe, ７５ Se, ７７ As, ８９ Sr, ９９ M.O. １０５ Rh, １０９ Pd, １４３ P.R. １４９ P.M., １６９ Er, １９４ Ir, １９８ Au, １９９ Au, １９９ Au and ２１１ Pb; and / or The diagnostic radionuclide is 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 l, 124 l, 125 l, 131 l, 142 Pr, 143 Pr, 149 Pm, selected from the group consisting of 153 Sm, 154″1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac; The anti-CLDN18.2 antibody conjugate of claim 1.

3. The anti-CLDN18.2 antibody conjugate of claim 1, wherein the diagnostic radionuclide is detectable by positron emission tomography (PET) or single photon emission computed tomography (SPECT).

4. The radionuclide is ６４ Cu, ６７ Cu, ８９ Zr 、１２４ I, ８６ Y. ９０ Y. １１１ In, １２３／１３１ I, １７７ Lu, １１ C. １４ C. ４１ Ca, ６７ G.A. ６８ G.A. １３ N. １５ O. ４４ Sc, １８ F. ９９ｍ Tc, and ９０ｍ The anti-CLDN18.2 antibody conjugate of claim 1, wherein the antibody is selected from the group consisting of Tc.

5. The anti-CLDN18.2 antibody conjugate of claim 2, wherein the radionuclide is labeled to the antibody or antigen-binding fragment thereof via a chelating agent.

6. The anti-CLDN18.2 antibody conjugate of claim 2, wherein the chelator contains three or more atoms for chelation, each atom being selected from the group consisting of nitrogen, sulfur, oxygen, and phosphorus.

7. The chelating agent is selected from the group consisting of DFO (delferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (NR-diethylenetriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-acetic acid), 1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid (abbreviated as TRITA in this specification); 7. The anti-CLDN18.2 antibody conjugate of claim 6, comprising tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (abbreviated herein as TETA); and 1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid (abbreviated herein as HETA), ethylenediaminetetraacetic acid (abbreviated herein as EDTA), or diethylenetriaminepentaacetic acid (DTPA).

8. The radionuclide ６４ Cu or ６７ Cu and the chelating agent comprises TETA, NOTA, NODA, or NODGA, or the radionuclide is ８９ 8. The antibody conjugate of claim 7, wherein the radionuclide is 124 I and the chelating agent comprises DFO, or the radionuclide is 124 I and the chelating agent comprises EDTA, or the radionuclide is 177 Lu and the chelating agent comprises DOTA.

9. The anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises the sequences of heavy chain HCDR1, HCDR2 and HCDR3, and / or light chain LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 sequence comprises GYNMN (SEQ ID NO:1), or TYFIGVG (SEQ ID NO:13), or a homologous sequence thereof with at least 80% sequence identity; The HCDR2 sequence is １ IDPYYX ２ X ３ T.X. ４ YNQKFX ５ G (SEQ ID NO:32), or HIWWNDNKYYNTALKS (SEQ ID NO:15), or a homologous sequence thereof with at least 80% sequence identity; The HCDR3 sequence is ６ X ７ X ８ GNAFDY (SEQ ID NO:33), or MGSGAWFTY (SEQ ID NO:17), or a homologous sequence thereof with at least 80% sequence identity; The LCDR1 sequence is KSSQX ９ LX １０ NX １１ GNX １２ KNYLT (SEQ ID NO:34), or a homologous sequence thereof with at least 80% sequence identity; The LCDR2 sequence is １３ S (SEQ ID NO:35), or a homologous sequence thereof with at least 80% sequence identity; The LCDR3 sequence is QNDYX １４ X １５ P.X. １６ T (SEQ ID NO:36), or a homologous sequence thereof with at least 80% sequence identity; Here, X １ is N or Y or H, and X ２ is G or V, and X ３ is A or G or T, and X ４ is R, T or S, and X ５ is K or R, and X ６ is S or M, and X ７ is Y or F, and X ８ is Y or H, and X ９ is S or N, and X １０ is L or F, and X １１ is S or N, and X １２ is Q or L, and X １３ is E or K, and X １４ is S or Y, and X １５ is F or Y, and X １６ is F or L. The antibody conjugate of claim 1 .

10. The anti-CLDN18.2 antibody or antigen-binding fragment thereof: a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:19, and an HCDR3 comprising the sequence of SEQ ID NO:21; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:16, and an LCDR3 comprising the sequence of SEQ ID NO:18; a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:3, and an HCDR3 comprising the sequence of SEQ ID NO:5; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:7, and an HCDR3 comprising the sequence of SEQ ID NO:5; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:8; a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:9, and an HCDR3 comprising the sequence of SEQ ID NO:11; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:10, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:13, an HCDR2 comprising the sequence of SEQ ID NO:15, and an HCDR3 comprising the sequence of SEQ ID NO:17; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:12; or a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:22, and an HCDR3 comprising the sequence of SEQ ID NO:5; and a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:20, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:

6. The antibody conjugate of claim 1 , comprising:

11. the heavy chain variable region comprises the sequence of SEQ ID NO:23 and the light chain variable region comprises the sequence of SEQ ID NO:24; the heavy chain variable region comprises the sequence of SEQ ID NO:25 and the light chain variable region comprises the sequence of SEQ ID NO:26; the heavy chain variable region comprises the sequence of SEQ ID NO:27 and the light chain variable region comprises the sequence of SEQ ID NO:28; the heavy chain variable region comprises the sequence of SEQ ID NO:29 and the light chain variable region comprises the sequence of SEQ ID NO:26 or 28; the heavy chain variable region comprises the sequence of SEQ ID NO:37 and the light chain variable region comprises the sequence of SEQ ID NO:38; the heavy chain variable region comprises the sequence of SEQ ID NO:39 and the light chain variable region comprises the sequence of SEQ ID NO:40; the heavy chain variable region comprises the sequence of SEQ ID NO:41 and the light chain variable region comprises the sequence of SEQ ID NO:42; the heavy chain variable region comprises the sequence of SEQ ID NO:43 and the light chain variable region comprises the sequence of SEQ ID NO:44; the heavy chain variable region comprises the sequence of SEQ ID NO:45 and the light chain variable region comprises the sequence of SEQ ID NO:46; or 2. The antibody conjugate of claim 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 47 and the light chain variable region comprises the sequence of SEQ ID NO:

48.

12. The antibody conjugate of claim 1, wherein the anti-CLDN18.2 antibody or antigen-binding fragment thereof further comprises a substitution or modification of one or more amino acid residues that still retains specific binding affinity to human CLDN18.2, and optionally the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a modification or substitution of one or more amino acid residues in its Fc region.

13. The antibody conjugate of claim 9, wherein the anti-CLDN18.2 antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region.

14. The antibody conjugate of claim 1, wherein the anti-CLDN18.2 antibody or antigen-binding fragment thereof is humanized.

15. The anti-CLDN18.2 antibody or antigen-binding fragment thereof is a diabody, Fab, Fab', F(ab') ２ , Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) ２ , bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv-Fc antibodies, scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies.

16. A pharmaceutical composition comprising the antibody conjugate of claim 1 and one or more pharma- ceutically acceptable carriers.

17. 13. A pharmaceutical composition comprising the antibody conjugate of claim 1 for use in a method for obtaining an image of a site of interest in a subject, the method comprising: a) administering to a subject an effective amount of the antibody conjugate of claim 1; b) subjecting the region of interest of the subject to Positron Emission Tomography (PET) or SPECT; c) identifying a detectable signal from a radionuclide in said subject; d) imaging the detectable signal, thereby obtaining an image of the site of interest in the subject; 13. A pharmaceutical composition comprising:

18. 13. A pharmaceutical composition comprising the antibody conjugate of claim 1 for use in a method of detecting or visualizing claudin18.2 expression in a subject in a non-invasive manner, the method comprising: a) administering to a subject an effective amount of the antibody conjugate of claim 1; b) subjecting the subject to Positron Emission Tomography (PET) or SPECT; c) identifying a detectable signal from the radionuclide at a site of interest in the subject; d) determining or visualizing claudin18.2 expression in said site of interest in said subject based on the identified detectable signal; 13. A pharmaceutical composition comprising:

19. 10. A pharmaceutical composition comprising the antibody conjugate of claim 1 for use in a method for monitoring, in a non-invasive manner, therapeutic efficacy, responsiveness to treatment, or the occurrence of resistance or recurrence, or metastasis, in a subject who has been treated for a therapeutic period, said method comprising: a) administering to a subject an effective amount of the antibody conjugate of claim 1; b) subjecting the subject to Positron Emission Tomography (PET) or SPECT; c) identifying a detectable signal from the radionuclide at a site of interest in the subject; d) determining post-treatment claudin 18.2 expression at the site of interest in said subject based on the identified detectable signal; and e) comparing the level or distribution of claudin18.2 expression after treatment to a baseline level or distribution of claudin18.2 expression obtained from said subject prior to a treatment period to determine a post-treatment change in said level or distribution of claudin18.2 expression in said subject; f) determining therapeutic efficacy, responsiveness to treatment, or occurrence of resistance or recurrence, or metastasis based on the changes determined in step (e); 13. A pharmaceutical composition comprising:

20. A pharmaceutical composition for treating or diagnosing a CLDN18.2-related disease or condition in a subject in need of such treatment, comprising an anti-CLDN18.2 antibody conjugate according to claim 1.

21. A kit comprising a first anti-CLDN18.2 antibody conjugate according to claim 1 and a second anti-CLDN18.2 antibody conjugate according to claim 1, wherein the first anti-CLDN18.2 antibody conjugate comprises a diagnostic radionuclide and the second anti-CLDN18.2 antibody conjugate comprises a therapeutic radionuclide.

22. An anti-CLDN18.2 antibody or an antigen-binding fragment thereof １２４ I, １２３ I or １３１ A method for preparing an anti-CLDN18.2 antibody conjugate according to claim 2, comprising reacting an iodide compound labeled with I in the presence of an enzymatic or chemical oxidizing agent.

23. conjugating an anti-CLDN18.2 antibody or antigen-binding fragment thereof with a chelating agent to obtain a chelating agent-antibody conjugate; ６４ Cu, 177 Lu or ８９ A method for preparing an anti-CLDN18.2 antibody conjugate according to claim 2, comprising reacting with Zr.

24. The chelating agent may be selected from the group consisting of DFO (delferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (NR-diethylenetriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-acetic acid), TRITA (1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid); TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid); and HETA (1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid), NETA ({4-[2-(bis-calcium phosphate) (1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid).

24. The method of claim 23, comprising the step of preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of: (a) preparing a tertiary amine compound comprising the steps of:

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