IL324747A - Anti-claudin18.2 antibody and method of making and using thereof - Google Patents

Description

EpiMED Docket No .: SIBA064PCT ANTI - CLAUDIN18.2 ANTIBODY AND METHOD OF MAKING AND USING THEREOF CROSS - REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application Ser . No. 63 / 468,198 filed May 22 , 2023 , under 35 U.S.C. 119 ( e ) , the entire disclosures of which are incorporated by reference herein . TECHNICAL FIELD The present disclosure generally relates to the technical field of antibody cancer therapeutics , and more particularly relates to anti - Claudin 18.2 antibodies . BACKGROUND Gastric and pancreatic cancers are among the most common malignancies worldwide . Gastric cancer is a major cause of death in many parts of Asia , South and Central America , and Eastern Europe . In 2020 , it was ranked the 5th most common cancer type with more than 1 million new cases ¹detroper . In the US , it was estimated that 26,380 cases would be diagnosed in 20and that death would occur in 11,090 of those cases . Between 2011 and 2017 , the combined overall - year survival rate was 32 % ² . Pancreatic cancer is the 12th most common cancer worldwide , with more than 495,000 cases reported in 2020³ . In the US , pancreatic cancer is more common than gastric cancer , accounting for 3 % of cancers and 7 % of cancer deaths4 . Due to the advanced stage of disease following diagnosis , the prognosis is poor with a combined 5 - year survival rate of only 11 % ² . While targeted therapeutics are improving , these malignancies represent an area of clearly unmet medical need . The overexpression of Claudin 18.2 ( aka , CLDN18.2 ) has been linked to several human malignancies , including gastric and pancreatic cancers . CLDN18.2 is a selective marker for gastric mucosal epithelial cells and , during normal expression , is buried within the tight junctions of healthy tissues . However , during carcinogenesis , the disruption of the epithelial cell barrier allows for the exposure of CLDN18.2 epitopes that can be accessed by targeted therapeutics5-7 . This distinction makes CLDN18.2 a unique , gastric cancer - specific target for immune therapies . In addition to gastric cancer , the overexpression of CLDN18.2 has been detected in several primary cancers , including breast , colon , esophageal , biliary tract , and pancreatic cancers . While CLDN18.2 is not detected in normal pancreatic tissues , in cases of primary pancreatic ductal adenocarcinoma , 59.2 % of cases had cells with CLDN18.2 positive ³noisserpxe . Several antibodies targeting CLDN18.2 for cancer immunotherapy are in development ; however , none have been approved for clinical ³esu . Therefore , a need remains for the development of highly specific anti - CLDN18.2 antibodies for treating malignancies such as gastric and pancreatic cancers . SUMMARY 1 EpiMED Docket No .: SIBA064PCT The disclosure generally relates to the technical field of antibody therapeutic agents , and more particularly relates to antibodies against specific epitopes of Claudin 18 , including CLDN18.and / or CLDN18.2 . In one aspect , the disclosure provides isolated monoclonal antibodies ( mAbs ) or antigen- binding fragments thereof having a binding specificity to CLDN18.1 , CLDN18.2 , or a combination thereof . In one embodiment , the mAbs or antigen - binding fragment thereof have a binding affinity to CLDN18.1 . In one embodiment , the mAbs or antigen - binding fragment thereof have a binding affinity to CLDN18.2 . In one embodiment , the mAbs or antigen - binding fragment thereof have a binding affinity to CLDN18.1 and CLDN 18.2 . In one embodiment , the isolated mAbs or antigen - binding fragment thereof comprise an amino acid sequence having at least 70 % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 2 , 4 , 6 , 8 , 10 , or 12. In one embodiment , the isolated mAbs or antigen - binding fragment thereof comprise an amino acid sequence having at least 70 % , 80 % , 85 % , 90 % , 95 % , % , 99 % , or 100 % identity with the SEQ ID NO : 14 or 16. In one embodiment , the isolated mAbs or antigen - binding fragment thereof comprise an amino acid sequence having at least 70 % , 80 % , % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 18 , 20 , 22 , 24 , 26 , or 28. In one embodiment , the isolated mAbs or antigen - binding fragment thereof comprise an amino acid sequence having at least 70 % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 30 , 32 , 34 , or 36. In one embodiment , the isolated mAbs or antigen - binding fragment thereof comprise an amino acid sequence having at least 70 % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 38 , 40 , 42 , 44 , 46 , 48 , 50 , or 52. In one embodiment , the isolated mAbs or antigen - binding fragment thereof comprise an amino acid sequence having at least 70 % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 54 or 56 . The expression of CLDN18.2 is associated with several human malignancies , including gastric and pancreatic cancers . The disclosure further discloses the development of anti- CLDN18.2 antibodies for treating cancer . In another aspect , the disclosure provides an isolated mAb or antigen - binding fragment thereof having a binding specificity to CLDN18 , comprising an amino acid sequence having at least 70 % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 2 , 4 , 6 , 8 , , 12 , 14 , 16 , 18 , 20 , 22 , 24 , 26 , 28 , 30 , 32 , 34 , 36 , 38 , 40 , 42 , 44 , 46 , 48 , 50 , 52 , 54 , or 56 . In one embodiment , the isolated mAb or antigen - binding fragment thereof selectively binds to CLDN18.2 but not CLDN18.1 . In one embodiment , isolated mAb or antigen - binding fragment thereof has a binding affinity to CLDN18.2 having a KD value of not more than 5 , 3 , 2 , 1.5 , 1.45 , 1.4 , 1 , or 0.8 nM . In one embodiment , isolated mAb or antigen - binding fragment thereof has a binding avidity to CLDN18.2 having a KD value of not more than 0.5 , 0.4 , 0.3 , 0.25 , 0.24 , 0.23 , 0.22 , 0.21 , 0.2 , or 0.18 nM . In one embodiment , the ratio of binding of the isolated mAb or antigen - binding fragment thereof to CLDN18.2 to CLDN18.1 is at least 4 , 5 , 8 , 10 , 15 , 20 , 30 , or 40. In one embodiment , the ratio of binding of the isolated mAb or antigen - binding fragment thereof to CLDN18.2 to CLDN18.1 is from about 4 to about 10 , from about 3 to about 12 , from about 10 to about 25 , or from about 12 to about 22 . 2 EpiMED Docket No .: SIBA064PCT In one embodiment , the isolated mAb or antigen - binding fragment further includes a human framework region . In one embodiment , the isolated mAb may be a humanized antibody , a chimeric antibody , or a recombinant antibody . In one embodiment , the isolated mAb is an IgG . In one embodiment , the isolated mAb may be a bispecific antibody , tri - specific antibody , or multi - specific antibody . In one embodiment , the antigen - binding fragment may include a Fv , a Fab , a F ( ab ' ) 2 , a scFv or a scFv2 fragment . In one embodiment , the disclosure provides an IgG1 heavy chain for the isolated mAb or antigen - binding fragment thereof , comprising an amino acid sequence having at least 70 % , 80 % , % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 2 , 6 , 10 , 14 , 18 , 22 , 26 , 30 , , 38 , 42 , 46 , 50 , or 54 . In one embodiment , the disclosure provides a kappa light chain for the isolated mAb or antigen - binding fragment as disclosed herein , comprising an amino acid sequence having at least % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity the SEQ ID NO : 4 , 8 , 12 , 16 , 20 , 24 , 28 , , 36 , 40 , 44 , 48 , 52 , or 56 . In one embodiment , the disclosure provides the variable light chain for the isolated mAb or antigen - binding fragment as disclosed herein , comprising an amino acid sequence having at least 70 % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 4 , 8 , 12 , 16 , , 24 , 28 , 32 , 36 , 40 , 44 , 48 , 52 , or 56 . In one embodiment , the disclosure provides the variable heavy chain for the isolated mAb or antigen - binding fragment as disclosed herein , comprising an amino acid sequence having at least 70 % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID NO : 2 , 6 , 10 , 14 , , 22 , 26 , 30 , 34 , 38 , 42 , 46 , 50 , or 54 . In one embodiment , the disclosure provides an isolated mAb or antigen - binding fragment thereof having a binding specificity to CLDN18 . In one embodiment , the isolated mAb or antigen- binding fragment thereof comprises a variable heavy chain and a variable light chain having complementarity determining region ( CDR ) H1 , CDRH2 , CDRH3 , CDRL1 , CDRL2 , and CDRLof SEQ ID NO : 57 , 58 , 59 , 60 , 61 , and 62 ; 63 , 64 , 65 , 66 , 67 , and 68 ; 69 , 70 , 71 , 72 , 73 , and 74 ; , 76 , 77 , 78 , 79 , and 80 ; or 81 , 82 , 83 , 84 , 85 , and 86 . In a further aspect , the disclosure provides an isolated nucleic acid encoding the isolated mAb or antigen - binding fragment , the IgG1 heavy Chain , the kappa light chain , the variable light chain , or the variable heavy chain , as disclosed herein . In one embodiment , the isolated nucleic acid has at least 70 % , 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , or 100 % identity with the SEQ ID . NO : 1 , , 5 , 7 , 9 , 11 , 13 , 15 , 17 , 19 , 21 , 23 , 25 , 27 , 29 , 31 , 33 , 35 , 37 , 39 , 41 , 43 , 45 , 47 , 49 , 51 , 53 , or 55 . In a further aspect , the disclosure provides an expression vector comprising the isolated nucleic acid as disclosed herein , wherein the vector is expressible in a cell . In a further aspect , the disclosure provides a host cell comprising the nucleic acid as disclosed herein , wherein the host cell is a prokaryotic cell or a eukaryotic cell . In a further aspect , the disclosure provides a method of producing an antibody comprising culturing the host cell as disclosed herein , so that the antibody is produced . 3 EpiMED Docket No .: SIBA064PCT In a further aspect , the disclosure provides an immuno - conjugate , comprising the isolated mAb or antigen - binding fragment thereof conjugated to a drug unit through a linker . In one embodiment , the linker comprises a covalent bond selected from an ester bond , an ether bond , an amine bond , an amide bond , a disulfide bond , an imide bond , a sulfone bond , a phosphate bond , a phosphorus ester bond , a peptide bond , a hydrazone bond or a combination thereof . In one embodiment , the drug unit comprises a cytotoxic agent , an immune regulatory reagent , or a combination thereof . In one embodiment , cytotoxic agent is selected from a growth inhibitory agent or a chemotherapeutic agent from a class of tubulin binders , DNA intercalators , DNA alkylators , enzyme inhibitors , immune modulators , antimetabolite agents , radioactive isotopesor a combination thereof . In one embodiment , the cytotoxic agent is selected from a calicheamicin , ozogamicin , monomethyl auristatin E , emtansinea derivative or a combination thereof . In one embodiment , the immune regulatory reagents activate or suppress immune cells , T cell , NK cell , B cell , macrophage , or dendritic cell . In a further aspect , the disclosure provides a pharmaceutical composition , comprising the isolated mAb or antigen - binding fragment thereof or the immuno - conjugate , and a pharmaceutically acceptable carrier . In one embodiment , the pharmaceutical composition further comprises a radioisotope , a radionuclide , a toxin , a therapeutic agent , a chemotherapeutic agent , or a combination thereof . In one embodiment , the therapeutic agent comprises an anti - estrogen agent , a receptor tyrosine kinase inhibitor , a kinase inhibitor , a cell cycle inhibitor , a DNA , RNA or protein synthesis inhibitor , a RAS inhibitor , or a combination thereof . In a further aspect , the disclosure provides a method of treating a subject with cancer . In one embodiment , the method comprises administering to the subject an effective amount of the isolated mAb or antigen - binding fragment thereof . In one embodiment , the method comprises administering to the subject an effective amount of the immuno - conjugate as disclosed herein . In one embodiment , the method comprises administering to the subject an effective amount of the pharmaceutical composition as disclosed herein . In one embodiment , the cancer comprises cells expressing Claudin 18.2 . In one embodiment , the cancer comprises colorectal cancer , pancreatic cancer , esophageal cancer , nasopharyngeal cancer , anal cancer , rectal cancer , gastric cancer , or bladder cancer . In one embodiment , the method of treating cancer further comprises co - administering an effective amount of a therapeutic agent . In one embodiment , the therapeutic agent comprises an antibody , a chemotherapy agent , an enzyme , anti - estrogen agent , a receptor tyrosine kinase inhibitor , a kinase inhibitor , a cell cycle inhibitor , a DNA , RNA or protein synthesis inhibitor , a RAS inhibitor or a combination thereof . In one embodiment , the subject is a human . In a further aspect , the disclosure provides a solution comprising an effective concentration of the isolated mAb or an antigen - binding fragment thereof , wherein the solution is blood plasma in a subject . In one embodiment , the disclosure provides a solution comprising an effective 4 EpiMED Docket No .: SIBA064PCT concentration of the immune - conjugate as disclosed herein . In one embodiment , the solution is blood plasma in the subject . BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other features of this disclosure may become more fully apparent from the following description and appended claims , taken in conjunction with the accompanying drawings . Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are , therefore , not to be considered limiting of its scope , the disclosure may be described with additional specificity and detail through use of the accompanying drawings , in which : Figure 1 shows the alignment of extracellular claudin domains ( ECD1 and ECD2 ) in the full- length amino acid sequences encoding human and cynomolgus monkey Claudin 18.1 and Claudin 18.2 ; Figure 2 shows the detection of anti - Claudin 18.2 antibodies in B cell culture screen by direct sandwich ELISA ; Figure 3 shows the detection of anti - Claudin 18.2 antibodies in B cell culture screen by flow cytometry using 293 HEK transfected with either a Claudin 18.2 or a Claudin 18.1 expression vector ; Figure 4 shows the characterization of chimeric anti - Claudin18.2 antibodies by flow cytometry using ExpiCHO cells transfected with either a Claudin 18.2 or a Claudin 18.1 expression vector ; Figure 5 shows the characterization of binding affinity of chimeric anti - Claudin 18.2 antibodies , 5208H10 ( 5A ) and 5103F3 ( 5B ) , by Bio - Layer Interferometry followed by fits and calculations using manufacturer - provided software followed by fits and calculations using manufacturer- provided software ; Figure 6 shows the characterization of binding avidity of chimeric anti - Claudin 18.2 antibodies by Bio - Layer Interferometry followed by fits and calculations using manufacturer - provided software ; Figure 7 shows the characterization of CLDN18.2 specificity of humanized anti - Claudin 18.antibody by flow cytometry and ExpiCHO cells transiently expressing BCMA - Claudin18 fusion proteins ; Figure 8 shows the characterization of CLDN18.2 specificity of humanized anti - Claudin 18.30 antibody by direct ELISA using recombinant protein VLPs ; Figure 9 shows the characterization of epitope binning of anti - Claudin 18.2 antibodies by Bio- Layer Interferometry ( OCTET ) ; Figure 10 shows the lysis of CLDN18.2 - exppressing CHO cells ( CHO - Claudin 18.2 ) in response to increasing doses of humanized anti - Claudin18.2 antibodies in Complement - Dependent Cytotoxicity ( CDC ) assay ; Figure 11 shows the lysis of Claudin 18.2 - exppressing CHO cells ( CHO - Claudin18.2 ) in the presence of donor NK cells as effector cells ( 5 : 1 to target cells ) and increasing doses of humanized anti - Claudin18.2 antibodies in Antibody - Dependent Cell - mediated Cytotoxicity ( ADCC ) assay ; Figure 12 shows the characterization of cross - specific interactions among anti - Claudin 18.antibodies of human , rat , and murine origins by flow cytometry ; EpiMED Docket No .: SIBA064PCT Figure 13 shows the characterization of lysosomal trafficking of anti - Claudin 18.2 antibodies at 5nM and 10nM concentrations by using time - series live - cell fluorescence microscopy ; and Figure 14 shows the sequence alignment between 5103F3 BSM and comparator antibodies ( 14A ) ; the % identities of all CDRs and framework only ( 14B , upper and lower panels , respectively ) , and the % identities of the entire variable region ( 14C ) . DETAILED DESCRIPTION The disclosure discloses the generation , characterization , and application of anti- CLDN18.2 antibodies for treating malignancies such as gastrointestinal tumors . In the following detailed description , reference is made to the accompanying drawings , which form a part hereof . In the drawings , similar symbols typically identify similar components , unless context dictates otherwise . The illustrative embodiments described in the detailed description , drawings , and claims are not meant to be limiting . Other embodiments may be utilized , and other changes may be made , without departing from the spirit or scope of the subject matter presented herein . It will be readily understood that the aspects of the present disclosure , as generally described herein , and illustrated in the Figures , can be arranged , substituted , combined , separated , and designed in a wide variety of different configurations , all of which are explicitly contemplated herein . Claudins ( aka , CLDNs ) are a family of proteins that have direct roles in the formation of tight junctions . Tight junctions form a paracellular barrier that controls the flow of molecules between cells . CLDNs are critical for the formation of these barriers and help to maintain the polarity of the epithelial cell layers8 . CLDNs are tetraspanin proteins composed of four transmembrane domains : N- and C - terminal ends within the cytoplasm , and two extracellular domain loops . CLDNs are expressed in normal tissues ; however , altered function and expression have been linked to the formation of cancer 5-6,8 . Of particular interest is Claudin 18.2 ( CLDN18.2 ) , which has been linked to several human malignancies , including gastric and pancreatic cancers 5-6 . The human CLDN18 gene contains 6 exons and 5 introns . Alternative splicing of exons 1a and lb forms two isoforms , CLDN18.1 and CLDN18.29 . These splice variants are restricted to different tissues , with CLDN18.1 predominantly expressed in normal lung tissue and CLDN18.expressed in gastric tissue 5-6 . The sequence of CLDN18.1 is almost identical to CLDN18.2 with only 8 amino acid differences within extracellular loop D1 ( ECDs in Figure 1 ) . It is this high sequence similarity that makes the discovery of CLDN18.2 - specific antibodies significantly more challenging . Several antibodies targeting CLDN18.2 for cancer immunotherapy are in development , including monospecific monoclonal antibodies ( mAbs ) , bispecific antibodies ( BsAbs ) , chimeric antigen receptor T cells ( CAR - T ) , and antibody - drug conjugates ( ADC ) . Zolbetuximab ( Astellas Pharma ) is a mouse chimeric mAb that specifically binds CLDN18.2 and has shown safety and efficacy for patients with HER2 - positive advanced gastric ³recnac . TST001 ( Mabspace Bio- Sciences ) is a humanized mAb that has shown comparable activity to Zolbetuximab . While the majority of antibodies under development are mAbs , BsAbs and ADCs have also proven to be valid methods of treatment for both gastric and pancreatic cancers , including AMG - 910 ( Amgen , 6 EpiMED Docket No .: SIBA064PCT bispecific T - cell engager ) and CMG901 ( Keymed Biosciences , ADC ) . However , no CLDN18.2- targeting antibodies have been approved for clinical application . The present disclosure provides , among others , isolated antibodies , methods of making such antibodies , monoclonal and / or recombinant monospecific antibodies , pharmaceutical compositions containing the antibodies , monoclonal and / or recombinant monospecific antibodies , the methods for making the antibodies and compositions , and the methods for treating cancer using the antibodies and compositions disclosed herein . Specifically , the present disclosure provides isolated monospecific monoclonal antibodies ( mAb ) or antigen - binding fragments thereof having a binding specificity to human Claudin18.2 , wherein the isolated mAb or antigen - binding fragments comprise an amino acid sequence having an identity with a sequence selected from SEQ ID NO . 2 , 4 , 7 , 8 , 10 , 12 , 14 , 16 , 18 , 20 , 22 , 24 , 26 , 28 , 30 , 32 , 34 , 36 , 38 , 40 , 42 , 44 , 46 , 48 , 50 , , 54 , or 56 . , ﻭ The terms " a " . " an ” and “ the ” as used herein are defined to mean “ one or more " and include the plural unless the context is inappropriate . The terms “ polypeptide " , " peptide ” , and “ protein ” , as used herein , are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond . The term " antigen " refers to an entity or fragment thereof which can induce an immune response in an organism , particularly an animal , more particularly a mammal including a human . The term includes immunogens and regions thereof responsible for antigenicity or antigenic determinants . The terms " antigen- or epitope - binding portion or fragment ” , “ variable region ” , “ variable region sequence ” , or “ binding domain ” refer to fragments of an antibody that are capable of binding to an antigen ( such as Claudin18.2 in this application ) . These fragments may be capable of the antigen - binding function and additional functions of the intact antibody . Examples of binding fragments include , but are not limited to , a single - chain Fv fragment ( scFv ) consisting of the variable light chain ( VL ) and variable heavy chain ( VH ) domains of a single arm of an antibody connected in a single polypeptide chain by a synthetic linker , or a Fab fragment which is a monovalent fragment consisting of the VL , constant light ( CL ) , VH and constant heavy 1 ( CH1 ) domains . Antibody fragments can be even smaller sub - fragments and can consist of domains as small as a single CDR domain , in particular , the CDR3 regions from either the VL and / or VH domains ( for example see Beiboer et al . , J. Mol . Biol . 296 : 833-49 ( 2000 ) ) . Antibody fragments are produced using conventional methods known to those skilled in the art . The antibody fragments can be screened for utility using the same techniques employed with intact antibodies . The “ antigen- or epitope - binding portion or fragment " , " variable region ” , “ variable region sequence ” , or “ binding domain ” may be derived from an antibody of the present disclosure by a number of art - known techniques . For example , purified monoclonal antibodies can be cleaved with an enzyme , such as pepsin , and subjected to HPLC gel filtration . Papain digestion of antibodies produces two identical antigen binding fragments , called " Fab " fragments , each with a single antigen binding site , and a residual “ Fc " fragment , whose name reflects its ability to crystallize readily . Pepsin treatment yields an F ( ab ' ) 2 fragment that has two antigen combining 7 EpiMED Docket No .: SIBA064PCT sites and is still capable of cross - linking antigen . The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like . For further description of general techniques for the isolation of active fragments of antibodies , see for example , Khaw , B. A. et al . J. Nucl . Med . 23 : 1011-1019 ( 1982 ) ; Rousseaux et al . Methods Enzymology , 121 : 663-69 , Academic Press , 1986 . The term " antibody " is used in the broadest sense and specifically covers single monoclonal antibodies and / or recombinant antibodies ( including agonist and antagonist antibodies ) , antibody compositions with polyepitope specificity , as well as antibody fragments ( e.g. , Fab , F ( ab ' ) 2 , and Fv ) , so long as they exhibit the desired biological activity . In some embodiments , the antibody may be monoclonal , polyclonal , chimeric , single chain , multi - specific or multi - effective , human and humanized antibodies , and / or active fragments thereof . Examples of active fragments of molecules that bind to known antigens include Fab , F ( ab ' ) 2 , scFv and Fv . fragments , including the products of a Fab immunoglobulin expression library and epitope - binding fragments of any of the antibodies and fragments mentioned above . The term " FV " refers to the minimum antibody fragment which contains a complete antigen recognition and binding site . This region consists of a dimer of one heavy and one light chain variable domain in tight , non - covalent association . It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH - VL dimer . Collectively , the six CDRs confer antigen binding specificity to the antibody . However , even a single variable domain ( or half of an Fv comprising only three CDRs specific for an antigen ) has the ability to recognize and bind antigen , although at a lower affinity than the entire binding site . In some embodiments , antibody may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules , i.e. , molecules that contain a binding site and that can immunospecifically bind to an antigen . A typical antibody refers to heterotetrameric protein comprising typically of two heavy ( H ) chains and two light ( L ) chains . Each heavy chain is comprised of a heavy chain variable domain ( abbreviated as VH ) and three heavy chain constant domains ( abbreviated as CH1 , CH2 , and CH3 ) . Each light chain is comprised of a light chain variable domain ( abbreviated as VL ) and a light chain constant domain ( abbreviated as CL ) . The light chains of antibodies ( immunoglobulins ) from any vertebrate species can be assigned to one of two clearly distinct types , called kappa and lambda , based on the amino acid sequences of their constant domains . The VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions ( CDRs ) , and more conserved regions called framework regions ( FR ) . Each variable domain ( either VH or VL ) is typically composed of three CDRs and four FRs , arranged in the following order : FR1 , CDR1 , FR2 , CDR2 , FR3 , CDR3 , FR4 from amino - terminus to carboxy - terminus . Within the variable regions of the light and heavy chains there are binding regions that interacts with the antigen . Depending on the amino acid sequence of the constant domain of their heavy chains , immunoglobulins can be assigned to different classes . There are five major classes of immunoglobulins : IgA , IgD , IgE , IgG and IgM , and several of these may be further divided into 8 EpiMED Docket No .: SIBA064PCT subclasses ( isotypes ) , e.g. , IgG1 , IgG2 , IgG3 , and IgG4 ; IgA1 and IgA2 . The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha , delta , epsilon , gamma , and mu , respectively . The subunit structures and three - dimensional configurations of different classes of immunoglobulins are well known . The term “ monoclonal antibody " as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies , i.e. , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts . Monoclonal antibodies are highly specific , being directed against a single antigenic site . Furthermore , in contrast to conventional ( polyclonal ) antibody preparations which typically include different antibodies directed against different determinants ( epitopes ) , each monoclonal antibody is directed against a single determinant on the antigen . In addition to their specificity , the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture , uncontaminated by other immunoglobulins . The modifier “ monoclonal ” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method . For example , the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler & Milstein , Nature , 256 : 495 ( 1975 ) , or may be made by recombinant DNA methods ( see , e.g. , U.S. Pat . No. 4,816,567 ) . " Recombinant " means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells . Monoclonal antibodies can be produced using various methods , including without limitation , mouse hybridoma , phage display , recombinant DNA , molecular cloning of antibodies directly from primary B cells , and antibody discovery methods ( see Siegel . Transfus . Clin . Biol . 2002 ; Tiller . New Biotechnol . 2011 ; Seeber et al . PLOS One . 2014 ) . Monoclonal antibodies may include “ chimeric ” antibodies ( immunoglobulins ) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass , while the remainder of the chain ( s ) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass , as well as fragments of such antibodies , so long as they exhibit the desired biological activity ( U.S. Pat . No. 4,816,567 ; and Morrison et al . , Proc . Natl . Acad . Sci . USA , 81 : 6851-6855 [ 1984 ] ) . The term " humanized antibody " refers to a type of engineered antibody having its CDRs derived from a non - human donor immunoglobulin , the remaining immunoglobulin - derived parts of the molecule being derived from one ( or more ) human immunoglobulin ( s ) . In addition , framework support residues may be altered to preserve binding affinity . Methods to obtain " humanized antibodies " are well known to those skilled in the art . [ see , e.g. , Queen et al . , Proc . Natl Acad Sci USA , 86 : 10029-10032 ( 1989 ) , Hodgson et al . , Bio / Technology , 9 : 421 ( 1991 ) ) . The terms " antigen- or epitope - binding portion or fragment " , " variable domain " , " variable region " , " variable region sequence " , or " binding domain ” refer to fragments of an antibody that are capable of binding to an antigen ( such as EGFR and HER3 in this application ) . The antigen- 9 EpiMED Docket No .: SIBA064PCT binding fragment ( Fab ) is a region ( Fab region ) on an antibody that binds to antigens . These fragments may be capable of the antigen - binding function and additional functions of the intact antibody . Examples of binding fragments include , but are not limited to , a single - chain Fv fragment ( scFv ) consisting of the variable light chain ( VL ) and variable heavy chain ( VH ) domains of a single arm of an antibody connected in a single polypeptide chain by a synthetic linker , or a Fab fragment which is a monovalent fragment consisting of the VL , constant light ( CL ) , VH and constant heavy 1 ( CH1 ) domain . The terms " antigen- or epitope - binding portion or fragment " , " variable region " , " variable region sequence " , or " binding domain ” may be derived from an antibody of the disclosure by several art - known techniques . For example , purified monoclonal antibodies can be cleaved with an enzyme , such as pepsin , and subjected to HPLC gel filtration . Papain digestion of antibodies produces two identical antigen binding fragments , called “ Fab " fragments , each with a single antigen binding site , and a residual “ Fc " fragment , whose name reflects its ability to crystallize readily . Pepsin treatment yields an F ( ab ' ) 2 fragment that has two antigen combining sites and is still capable of cross - linking antigen . The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like . For further description of general techniques for the isolation of active fragments of antibodies ( see for example , Khaw , B. A. et al . J. Nucl . Med . 23 : 1011-1019 ( 1982 ) ; Rousseaux et al . Methods Enzymology , 121 : 663-69 , Academic Press , 1986 ) . The term “ Octet ” refers Bio - Layer Interferometry ( BLI ) , a label - free method for measuring bi - molecular interactions . The Dip and Read technology eliminate the need of microfluidics and means there is no need to purify antibodies to get valuable antigen binding and specificity information . The present disclosure may be understood more readily by reference to the following detailed description of specific embodiments and examples included herein . Although the present disclosure has been described with reference to specific details of certain embodiments thereof , it is not intended that such details should be regarded as limitations upon the scope of the disclosure . EXAMPLES Example 1. Generation of rabbit antibodies to Claudin18 : Immunizations : CLDN18.2 - specific antibodies were obtained by immunization of New Zealand white rabbits . Rabbits were split into two cohorts . Cohort 1 received a prime immunization with 1x1Claudin 18.2 - transfected 293 Human Embryonic Kidney ( HEK ) cells in Complete Freund's adjuvant . 293 HEK cells were transfected with a eukaryotic expression vector encoding human full - length CLDN18.2 ( Figure 1 , SEQ ID NO . 87 ) . Following the Immunization Protocol in Table 1 , rabbits then received 4 boosts at 7 - day intervals containing 2 - fold decreasing cell numbers in alternating Incomplete Freund's adjuvant and Alum CpG2007 . After the 4th immunization , rabbits received 7 boosts at 21 - day intervals . All boosts contained 2 - fold decreasing cell numbers in alternating Incomplete Freund's adjuvant and Alum CpG2007 , except for days 49 and 70 , where rabbits received 25 gµ of recombinant human Claudin 18.2 assembled into virus - like particles EpiMED Docket No .: SIBA064PCT ( VLPs ) ( Kactus Biosystems Cat . No. CLD - HE1822 ) . The same immunization strategy was used for Cohort 2 , except rabbits received 10 - fold higher cell numbers and 50 gµ recombinant human Claudin 18.2 VLP . For both cohorts , whole peripheral blood was collected three weeks after each boost , and PBMC were isolated and stored in liquid nitrogen for sorting . PBMC Isolation : Diluted blood was layered onto Lympholyte Cell Separation Media ( Cedarlane , Cat . No. CL5050 ) and centrifuged at 2500 rpm for 30 minutes . Following centrifugation , the PBMC layer was carefully aspirated and washed . RBC remaining within the pellet were lysed using RBC Lysis Buffer ( Qiagen , Cat . No. 158904 ) . After lysis , cells were washed and resuspended in 10 % DMSO in FBS at a cell concentration of 701x5≤ cells per ml . Isolation of Rabbit B - Cells by FACS : PBMC were thawed in immune cell media ( ICM ) , then incubated with an antibody staining mix containing recombinant human CLDN18.2 VLP and reagents specific to rabbit B - cell and T- cell markers . Following incubation , PBMC were washed and incubated with fluorophore - specific microbeads . PBMC were enriched for B - cells by magnetic - activated cell sorting ( MACS ) using a QuadroMACS Separator magnet ( Miltenyi Biotec , Cat . No. 130-090-976 ) . Cells were then stained with efluor780 viability dye ( ebioscience , Cat . No. 65-0865-14 ) , washed , resuspended in buffer , and stored at 4C until sorting by Fluorescence Activated Cell Sorting ( FACS ) . To sort B - cells , the appropriate compensation was performed using both sorter software and manual adjustments . Gates were adjusted to obtain live , IgG - positive rabbit B - cells . B - cells were sorted from the final gate as single cells into 96 - well plates containing previously prepared B - cell culture media containing ICM , rabbit splenocyte conditioned media , and feeder cells . For each sort , up to 30 plates were sorted . Plates were incubated at 37 ° C with 5 % CO2 for 12 days prior to screening . Example 2. Identification of anti - Claudin 18.2 antibodies : Antibody Screen by ELISA : B - cell cultures were screened for IgG secretion and Claudin 18.2 binding by direct sandwich ELISA . Plates were coated with 1 gμ / ml anti - rabbit IgG Fc fragment ( Jackson ImmunoResearch , Cat . No. 111-005-046 ) or Claudin 18.2 VLP ( Kactus Biosystems Cat . No. CLD- HE1822 ) . Plates were washed three times with 1X PBST , then blocked with 2 % BSA in DPBS . B - cell supernatant was harvested from culture plates and used for screening . B - cells remaining in the culture flask were stored in RNA Later ( Invitrogen , Cat . No. AM7021 ) at -80 ° C for RT - PCR amplification of antibody variable regions . After blocking , plates were washed as before , and B- cell supernatant was added to each plate at a 1:10 dilution . Following 1 hour incubation , plates were washed as before , then incubated with anti - Rb IgG secondary antibody conjugated to HRP ( Jackson ImmunoResearch , Cat . No. 111-035-046 ) for 30 minutes . Plates were washed , then incubated with TMB substrate ( ThermoFisher Scientific , Cat . No. 34029 ) . Reactions were stopped by adding commercial stop solution ( ThermoFisher Scientific , Cat . No. SS04 ) and plates were read using a spectrophotometer at 450 nm . Detection of rabbit IgG antibodies specific to Claudin18.were confirmed based off OD values exceeding the secondary only negative control . 11 EpiMED Docket No .: SIBA064PCT The rabbit IgG antibodies were grouped and plotted in Figure 2. For Sort # 1 , 17.4 % of B- cells sorted were IgG - positive . A total of 11 clones bound specifically to Claudin 18.2 VLP . For Sort # 2 , 28.5 % of B - cells sorted were IgG - positive . A total of 24 clones bound specifically to Claudin 18.2 VLP . For Sort # 3 , 60 % of B - cells sorted were IgG - positive . A total of 54 clones bound specifically to Claudin 18.2 VLP . Antibody Screen by Flow Cytometry : To confirm specificity , clones were tested for binding to Claudin18.2 and Claudin18.expressed on the surface of transfected 293 HEK . Detection of antibody binding to surface expressed Claudin 18.2 was achieved by flow cytometry . 293 HEK cells were transiently transfected with eukaryotic expression vectors encoding human full - length Claudin 18.2 and Claudin 18.1 ( Figure 1 , SEQ ID NO . 87 and 88 , respectively ) . 293 HEK transfected with irrelevant DNA were used as a negative control . Two days following transfection , cells were collected , stained with cell tracker dye ( ThermoFisher Scientific , Cat . No. C34565 ) and plated at ~ 1.5x1cells per well . B - cell supernatant was added to the cells at a 1 : 5 dilution and incubated for 1 hour at 4 ° C . Cells were washed , then incubated with Alexa Fluor 647 conjugated anti - rabbit IgG Fc secondary antibody ( Jackson ImmunoResearch , Cat . No. 111-605-008 ) for 30 minutes at 4 ° C . After one wash , cells were resuspended in FACS Buffer . Binding was determined by flow cytometry using a BD Fortessa flow cytometer . Clones expressing antibodies that bound Claudin 18.2 , but not Claudin 18.1 or negative control , were selected for molecular “ rescue ” of the antibody variable regions . To identify anti - Claudin 18.2 antibodies , B - cell clones that bound surface expressed Claudin 18.1 and Claudin 18.2 , but not the negative control , were selected for further screening . Representative clones bound Claudin 18.2 , but not Claudin 18.1 . The binding activity of B - cell supernatants obtained from Sorts # 2 and # 3 are shown ( Figure 3 ) . One clone , which bound both Claudin 18.1 and Claudin18.2 , was expressed specifically for use as a screening reagent ( 5208H10 ) . The disclosed antibodies selected are identified with black arrows ( Sort # 2 : 5103F3 , Sort # 3 : 5208H10 ) . Example 3. Production of rabbit / human chimeric monoclonal antibodies : Amplification of Antibody Variable Regions : B - cell clones identified by ELISA and flow cytometry were thawed from storage at -80 ° C . Heavy and light chain variable sequences were amplified by multiplex RT - PCR using primers specific to the leader sequence and constant region of rabbit IgG and rabbit kappa sequences . Amplicons were further amplified in a secondary PCR using nested primers that contained restriction sites . Heavy chain and light chain amplicons were cloned into an expression vector containing human IgG1 or human IgK , respectively . After sequence verification , expression plasmids were transiently co - transfected into 293 HEK cells to produce rabbit / human chimeric antibodies . Characterization of Chimeric Anti - Claudin 18.2 Antibodies - Flow Cytometry : Recombinant antibody supernatants were screened for binding to surface expressed Claudin 18.2 by flow cytometry . ExpiCHO cells were transiently transfected with eukaryotic 12 EpiMED Docket No .: SIBA064PCT expression vectors encoding human full - length Claudin 18.2 and Claudin 18.1 ( SEQ ID NO . 87 and , respectively ) . ExpiCHO cells transfected with irrelevant DNA were used as a negative control . Two days following transfection , cells were stained for flow cytometry as described for B - cell culture , however cells were incubated with chimeric antibodies serially diluted from 10 gµ / ml to 0.0625 ug / ml . As shown in Figure 4 , representative antibodies will bind Claudin18.2 at high MFI but will not bind Claudin18.1 or the negative control . All chimeric antibodies bound to Claudin18.2 . Claudin 18.1 binding was not detected for all antibodies except 5208H10 . 5208H10 , a pan- Claudin 18 binder , serves as a control for Claudin 18.1 expression . Detection of both Claudin18.and Claudin 18.2 on transiently transfected ExpiCHO supports the reported specificity of the anti- Claudin 18.2 clones to Claudin 18.2 . - Characterization of Chimeric Anti - Claudin18.2 Antibodies – OCTET Affinity : Recombinant antibody supernatants were validated for Claudin18.2 binding by bio - layer interferometry ( also known as OCTET ) using a ForteBio Octet Red 384 instrument . Antibodies ( 10 gµ / ml ) were captured onto anti - human Fc biosensors , then associated with recombinant human Claudin 18.2 - His full - length protein ( Acro Biosystems , Cat . No. CL2 - H82E3 ) diluted 1 : 3 from 3nM . Sensors were moved to wells containing kinetics buffer , and the dissociation of the antibody from Claudin18.2 was measured . Curve fits and affinity calculations were analyzed using manufacturer - provided software . Chimeric antibodies bound recombinant human Claudin 18.2 by bio - layer interferometry at high affinity with KD values in 1 to 3 nM range as shown in Figure 5 . Characterization of Chimeric Anti - Claudin 18.2 Antibodies – OCTET Avidity : Bio - layer interferometry ( i.e. , OCTET ) was also used to evaluate chimeric antibody avidity to recombinant human Claudin18.2 . Biotinylated recombinant human Claudin 18.2 ( 10 gµ / ml ) was captured onto streptavidin biosensors , then associated with chimeric antibodies diluted 1 : 3 from 300 nM . Sensors were moved to wells containing kinetics buffer to analyze dissociation of the antibody / Claudin 18.2 complex . Curve fits and avidity calculations were analyzed using manufacturer - provided software . The avidity for the chimeric antibody to recombinant human Claudin 18.2 was within 1 to 2 nM range as shown in Figure 6. Due to being only a control reagent , 5208H10 was not evaluated for avidity . Example 4. Production of humanized monoclonal antibodies : Humanization of Heavy and Light Chain Variable Regions : The heavy and light chain variable regions for antibodies were humanized using the ' Predict Humanizing Mutations ' algorithm in Discovery Studio . Methods chosen for humanization included best single mutation ( BSM ) , frequent residue substitution ( FRS ) , or germline graft ( GS ) . DNA sequences encoding the humanized antibody VH and VL regions were cloned into mammalian expression vectors containing human IgG1 / kappa constant regions using gene fragments ( Genewiz ) . Due to its use as a research tool , the pan - Claudin 18 binder ( 5208H10 ) was not humanized . Protein Stability by UPLC - SEC : 13 EpiMED Docket No .: SIBA064PCT Immediately after first - step protein A purification , antibodies were analyzed by analytical SEC using Waters Acquity UPLC H - Class with ACQUITY ®CLPU Protein BEH SEC Å002 , 4.6mm x 150mm , 1.7 mμ column . PBS ( 125 mM sodium phosphate , 137 mM sodium chloride , pH 6.8 ) was used as mobile phase for 10 - minute runs at 0.3 ml / min , injecting 10 gµ protein . Proteins were buffer - exchanged into 25 mM sodium acetate , 125 mM NaCl , 10 % sucrose , pH 5.5 . Antibody samples were injected into the same column after approximately three weeks and six weeks after the initial purification to test stability over time . UPLC - SEC traces were integrated into three categories : % POI ( protein of interest ) , % HMW ( high molecular weight ) , and % LMW ( low molecular weight ) . The change in each species ( POI , HMW , LMW ) was calculated based on the difference in the integrated area between the first and last stability time points . As summarized in Table 2 , the data demonstrate that most humanized anti - Claudin 18.2 antibodies were stable for at least four to six weeks under these conditions , showing negligible change in the protein of interest species over time . Example 5. Characterization of humanized antibodies : Flow Cytometry and OCTET : Humanized antibodies were screened by Flow Cytometry and OCTET as described earlier . For comparison , anti - CLDN18.2 antibodies included : the humanized antibody , 5103F3 BSM ; the pan - Claudin18 binder antibody , 5208H10 ( SI - 64CH5 ) ; Zolbetuximab ( SI - 64C8 ) ; and other positive control antibodies , SI - 64C9 , SI - 64C10 , and SI - 64C11 . Specifically , the anti - CLDN18.antibodies were expressed by transiently transfected ExpiCHO and the levels of expression were scored ( Table 3 ) . To evaluate the binding affinity and avidity of anti - CLDN antibodies , the Bio- Layer Interferometry was used to measure KD values as shown in Table 4. The KD values of anti - CLDN18.2 antibodies were clustered between 0.7 nM and 1.45 nM or within a range of 2- fold for affinity , whereas the KD values for avidity were between 0.22 nM and 5.31 nM or a more than 20X improvement in functional affinity of 5103F3 BSM over Zolbetuximab . Validation of Claudin 18.2 Specificity by Flow Cytometry : To further confirm the specificity of representative antibodies to Claudin18.2 and not Claudin 18.1 , BCMA - Claudin18 fusion proteins were expressed in transiently transfected ExpiCHO . Flow cytometry methods were followed as described previously . BCMA expression was detected using an anti - BCMA antibody . Detection of BCMA confirmed expression of BCMA - Claudin 18.1 and BCMA - Claudin 18.2 as shown in Figure 7. Expression was also confirmed using the pan - Claudin 18 binder , 5208H10 . Both controls confirmed that lack of binding by humanized antibodies was not due to lack of expression . Clone 5103F3 bound BCMA- Claudin 18.2 , but not BCMA - Claudin 18.1 , further supporting specificity to Claudin18.2 . Validation of Claudin 18.2 Specificity by ELISA : Specificity of representative antibody binding to Claudin 18.2 was further validated by direct sandwich ELISA as previously described , with the following changes . Plates were coated with 1 gµ / ml recombinant protein VLPs : Claudin18.2 VLP ( Acro Biosystems , Cat . No. CL2- H52P7 ) , Claudin 18.1 VLP ( Acro Biosystems , Cat . No. CL1 - H52P3 ) , or empty VLP isotype control ( Acro Biosystems , Cat . No. VLP - N5213 ) . Antibodies were tested at 10 gµ / ml , and binding 14 EpiMED Docket No .: SIBA064PCT was detected using an anti - Hu IgG secondary antibody conjugated to HRP ( Jackson ImmunoResearch , Cat . No. 309-035-003 ) . The result shows that 5103F3 BSM bound CLDN18.2- VLP , but not CLDN18.1 - VLP or VLP - only control ( Figure 8 ) . The pan - Claudin 18 binder , 5208H8 , bound both CLDN18.1 and CLDN18.2 , but not VLP - only control . Taken together , the flow cytometry and ELISA data demonstrate the specificity of 5103F3 BSM to Claudin 18.2 . Antibody Binning by Bio - Layer Interferometry ( OCTET ) : The epitopes of antibodies were binned by Bio - Layer Interferometry ( i.e. , OCTET ) In- Tandem Assay using a ForteBio OCTET Red 384 instrument . Biotinylated recombinant human Claudin 18.2 - His full - length protein ( Acro Biosystems , Cat . No. CL2 - H82E3 , 10 gµ / ml ) was captured onto streptavidin - conjugated biosensors , then associated with 10 gµ / ml of antibody ( i.e. , 1st association ) . Sensors were then moved to wells containing a different antibody ( i.e. , 2nd association ) . Using the instrument software , antibodies were binned based off hierarchical clustering of the change in response from the 1st association to the 2nd association . To determine blocking or nonblocking antibody pairs , a threshold was set at the appropriate self - binding signal . Antibodies that blocked binding were clustered into the same bin and considered to have similar epitopes . Antibodies that did not block binding were clustered into separate bins and considered to have different epitopes . The hierarchical clustering of disclosed antibodies , candidate antibodies , and positive control comparator antibodies ( SI - 64C8 to SI - 64C11 ) are shown in the binning matrix table ( Figure 9 ) . Multiple candidate antibodies blocked binding of the comparator antibodies , indicating that these clones have similar epitopes to those that are already in development . However , the disclosed clone , 5103F3 BSM , showed unidirectional binding activity to all antibodies , including most comparator antibodies . When bound to Claudin 18.2 as Antibody # 1 , 5103F3 BSM did not block binding of the candidate or comparator antibodies , except SI - 64C8 . When bound as Antibody # 2 , 5103F3 BSM was blocked by all antibodies . After clustering , 5103F3 BSM was organized into a bin that was distinguishable from all candidate and comparator antibodies ( circle ) . This suggests that 5103F3 BSM has a unique epitope - blocking profile when compared to other blocking antibodies .

Example 6. Anti - Claudin 18.2 antibody - dependent cytotoxicity : Complement - Dependent Cytotoxicity ( CDC ) : The Fc - mediated CDC activity of antibody - based therapeutics can be compared in vitro to determine if there are features of an antibody that enable a greater potential therapeutics mode of action . To compare the CDC function of 5103F3 BSM and Zolbetuximab , Chinese hamster ovarian cells ( CHO ) were transduced to express Claudin 18.2 tagged to a fluorescent reporter . Target cells CHO - Claudin 18.2 were treated with 5103F3 BSM and Zolbetuximab in the presence and absence of Normal Human Serum ( NHS ) to evaluate CDC in 2 hours . Cells were treated with antibody in the assay ranging from 6.4 pM to 500 nM across a 5 - fold dilution curve . CDC was evaluated by uptake of membrane impermeant fluorescent dye measured using FACS .

EpiMED Docket No .: SIBA064PCT The CDC activity of Zolbetuximab was detectable in this assay system . 5103F3 BSM showed equivalent CDC activity . This is consistent with equivalent binding activity over the duration of the assay and access to assemble the CDC multimeric compliment cascade . Antibody - dependent Cell - mediated Cytotoxicity ( ADCC ) : The Fc - mediated ADCC activity of antibody - based therapeutics can be compared in vitro to determine if there are features of an antibody that enable a greater potential therapeutics mode of action . In this testing system , a low ADCC responses may be due to the available antigen expression level over the duration of the assay . As a result , different binding properties , or antibody internalization may have an impact on the ADCC activity 10 . To test this , human primary NK ( Natural Killer ) cells were enriched from whole blood by negative selection , followed by density gradient centrifugation to become “ effector ” cells . The CHO cells that were transduced to express Claudin 18.2 tagged to a fluorescent reporter were target cells . The CHO - Claudin 18.2 target cells were treated with 5103F3 BSM , Zolbetuximab or rituximab , ranging from 100 nM to 0.00001 nM in a 10 - fold serial dilution . NK cells were added at a 5 : 1 E : T ( effector : target ) ratio to evaluate ADCC within the 24 - hour assay timeframe . ADCC was evaluated by loss of CHO - Claudin 18.2 cell reporter signal using time - series fluorescent microscopy . The ADCC activity of Zolbetuximab was detectable in this assay system and the CDspecific Rituximab did not demonstrate ADCC activity . 5103F3 BSM showed significantly different pharmacokinetics characterized by extending the ADCC activity . The enhanced ADCC activity of 5103F3 BSM compared to Zolbetuximab is consistent with greater antigen availability over the course of the testing allowing for greater NK cell engagement and cytolytic function . Example 7. Internalization of anti - CLDN18.2 antibodies and lysosomal trafficking : For many antibody - drug conjugate ( ADC ) drugs , a key step of the mechanism of action involves internalization " . Following antibody binding , internalization can facilitate the uptake of cytotoxic payload and trafficking to cancer cell lysosomes where lysosomes act as recycling centers within cells . Depending on the types of cytotoxic payload , cell lysis follows different mechanisms of action . Claudin 18.2 is a tight junction protein normally expressed on gastric epithelial cells and has additionally been detected in gastric , pancreatic , biliary tract and colorectal cancers 12 Thus , the mechanism of internalization and trafficking to lysosomes by anti- Claudin 18.2 may be the same , but the efficacy may depend on the antibody binding . The anti - Claudin 18.2 antibodies , 5103F3 BSM and Zolbetuximab , were labeled with FabFluor - pH red , an acid - sensitive fluorescent reagent that allows quantification of the trafficking . of internalized proteins to acidic lysosomes . The fluorescent signal of FabFluor - pH red is dependent on the low pH environment of lysosomes . In this way , the accumulation of internalized antibodies into lysosomal compartments may be quantified over time using time - series fluorescent microscopy . NUGC - 4 cell line was thawed and cultured in RPMI media supplemented with Pen / Strep and 10 % FBS . Cells were seeded at 10,000 cells / well in 50 Lμ of culture in 96 - well round bottom ULA plates ( Corning ) . Vehicle buffer , 5103F3 BSM , and Zolbetuximab were labeled with FabFluor - pH according to the manufacturer's instructions . Fifty microliters of 16 EpiMED Docket No .: SIBA064PCT FabFluor - pH labeled antibodies or control treatments were added to plated cells at 2X ( 10 nM or 20nM ) concentrations for 100 lµμ final assay volume / well . Cells were cultured in an Incucyte Slive cell imager ( Sartorius ) in a 37 ° C , 5 % CO2 humidified incubator for 16 hours . Using time - series live - cell fluorescence microscopy , the internalization and trafficking signals were readily detectable for both 5103F3 BSM and Zolbetuximab at either 5nM or 10nM concentrations . The signals were automatically counted and plotted over time . The result shows that 5103F3 BSM was internalized more efficiently and trafficked more signals ( indicative of cytotoxic payload in case of ADC ) to lysosomes than Zolbetuximab ( Figure 13 ) . Example 8. Characterization of 5103F3 BSM : Cross - Species Binding to Mouse and Rat Claudin18.2 : Humanized anti - Claudin 18.2 antibodies were analyzed for cross - species binding to mouse and rat Claudin18 by flow cytometry . ExpiCHO were transiently transfected with plasmids . expressing mouse or rat Claudin 18.1 and Claudin 18.2 . Binding of surface - expressed Claudin was determined using previously described methods . Table 5 shows that the antibody 5103Fbound mouse and rat Claudin 18.2 , but not Claudin 18.1 . Due to high sequence similarity to human ( 98.9 % for both extracellular domains ) , cynomolgus monkey Claudin 18 was not included in the assay . The binding of 5103F3 BSM to mouse Claudin 18.2 was confirmed by OCTET , with a binding affinity of 1.73 nM ( Figure 13 ) . Zolbetuximab is a human / mouse chimeric antibody therapeutics for treating patients with gastric cancer in combination with chemotherapy . In contrast , 5103F3 BSM is a humanized antibody . With a higher degree of humanness , 5103F3 BSM may exert improved therapeutic effect by reducing immunogenicity or other unwanted side effects after administration . In addition , 5103F3 BSM recognized a distinct epitope bin from isoform 2 of Claudin - 18 when compared to Zolbetuximab ( SI - 64C8 ) and other anti - CLDN18.2 antibodies , including AB011 ( SI - 64C 10 ) , LM- 102 ( SI - 64C9 ) , and MIL93 ( SI - 64C11 ) ( Figure 9 ) . This binding character of 5103F3 BSM may lead to unique binding specificity , clustering , internalization , and other biological activities . Indeed , 5103F3 BSM and Zolbetuximab have exhibited similar CDC but different ADCC activities ( Figure 10 and 11 ) , as well as different pharmacokinetics in internalization and lysosomal trafficking ( Figure 12 ) . Sequence Alignment of Lead and Comparator Antibodies : To investigate the structural basis of 5103F3 BSM's unique characters , the coding sequences 5103F3 BSM , 5208H10 , and comparator antibodies were aligned in the Kabat numbering scheme using Geneious Prime bioinformatics software and shown in Figure 14. The complementarity - determining regions ( CDRs ) of heavy and light chains were aligned across all antibodies ( boxed in upper and lower panels of Figure 14A ) . The percentage identity for all CDRs only , the framework regions only , and the entire variable region were calculated for comparison in Figure 14B and 14C . Of aligned CDR sequences , 5103F3 BSM shared 17-28 % identity with each comparator whereas each comparator shared 50-82 % identify with another comparator . Of all framework regions , 5103F3 BSM shared up to 70 % identity with each comparator . Using a two - tailed , one - sample T - test , the percentage identity of 5103F3 BSM to a given comparator was 17 EpiMED Docket No .: SIBA064PCT then compared to the average of percentage identities among the remaining comparators . The differences were significant ( P < 0.05 ) in both CDRs and the full variable regions but not in all framework regions ( P > 0.05 ) ( Table 6 ) . The significant differences in the amino acid sequences encoding the primary structure of CDRs underlie the characteristic features of 5103F3 BSM as compared to comparator antibodies in epitope binding ( Figure 9 ) and consequently , the biological activity . In summary , 5103F3 BSM is a humanized anti - CLDN18.2 antibody with high affinity , avidity , and specificity to Claudin 18.2 , whereas 5208H10 is a pan - Claudin 18 binder capable of binding both Claudin 18.1 and Claudin 18.2 . The pair of antibodies are useful for developing antibody therapy as well as diagnosing Claudin expression in different forms of cancer . Additionally , due to the significant technical advantages of 5103F3 BSM over other antibodies in the similar category including its high selectivity of Claudin 18.2 , 5103F3 BSM provides significant therapeutical advantage for treating cancers with overexpression of Claudin 18.2 . 18 EpiMED Docket No .: SIBA064PCT Reference 1. Stomach cancer statistics | WCRF International ( https://www.wcrf.org/cancer- trends / stomach - cancer - statistics / ) 2. American Cancer Society : Cancer Facts and Figures 2022. American Cancer Society , 2022. ( Cancer Facts & Figures 2022 ) 3. Pancreatic cancer statistics - WCRF International ( https://www.wcrf.org/cancer- trends / pancreatic - cancer - statistics / ) 4. Key Statistics for Pancreatic Cancer ( https://www.cancer.org/cancer/pancreatic- cancer / about / key - statistics.html ) 5. Cao W , Xing H , Li Y , Tian W , Song Y , Jiang Z , Yu J. Claudin 18.2 is a novel molecular biomarker for tumor - targeted immunotherapy . Biomark Res . 2022 May 31 ; 10 ( 1 ) : 38 . doi : 10.1186 / s40364-022-00385-1 . PMID : 35642043 ; PMCID : PMC9153115 . 6. Sahin U , Koslowski M , Dhaene K , Usener D , Brandenburg G , Seitz G , Huber C , icerüT 0. Claudin - 18 splice variant 2 is a pan - cancer target suitable for therapeutic antibody development . Clin Cancer Res . 2008 Dec 1 ; 14 ( 23 ) : 7624-34 . doi : 10.1158 / 1078- 0432.CCR - 08-1547 . PMID : 19047087 . 7. Niimi T , Nagashima K , Ward JM , Minoo P , Zimonjic DB , Popescu NC , Kimura S. 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 . Mol Cell Biol . 2001 Nov ; 21 ( 21 ) : 7380-90 . doi : 10.1128 / MCB.21.21.7380- 7390.2001 . PMID : 11585919 ; PMCID : PMC99911 . 8. Li J. Targeting claudins in cancer : diagnosis , prognosis and therapy . Am J Cancer Res . 2021 Jul 15 ; 11 ( 7 ) : 3406-3424 . PMID : 34354852 ; PMCID : PMC8332862 . 9. icerüT O , Koslowski M , Helftenbein G , Castle J , Rohde C , Dhaene K , Seitz G , Sahin U. Claudin - 18 gene structure , regulation , and expression is evolutionary conserved in mammals . Gene . 2011 Aug 1 ; 481 ( 2 ) : 83-92 . doi : 10.1016 / j.gene.2011.04.007 . Epub 20May 4. PMID : 21571049 . 10. Garvin D , Stecha P , Gilden J , Wang J , Grailer J , Hartnett J , Fan F , Cong M , Cheng ZJ . Determining ADCC Activity of Antibody - Based Therapeutic Molecules using Two Bioluminescent Reporter - Based Bioassays . Curr Protoc . 2021 Nov ; 1 ( 11 ) : e296 . 11. Yu et al . Frontiers Molecular Biosciences 2022 . 12. Yong Hong et al . Trans Cancer Res 2020 . 19 EpiMED Docket No .: SIBA064PCT TABLES Table 1. Immunization protocol for generating anti - CLDN18.2 antibody - Cohort 1 and Dose for Day Post- Immunization Immunogen Cohort ( gμ or cell no . ) Dose for Cohort ( gµ or cell no . ) Whole Adjuvant Bleed 293HEK- Complete 1x107 cells 1x108 cells No CLDN18.2 Freund's 293HEK- Incomplete 1x107 cells 1x108 cells No CLDN18.2 Freund's 293HEK- Alum 5x106 cells 5x107 cells No CLDN18.CpG20293HEK- Incomplete 5x106 cells 5x107 cells No CLDN18.2 Freund's 293HEK- Alum 2.5x106 cells 2.5x107 cells Yes CLDN18.CpG20Rec . Hu Incomplete gμ 50 gμ Yes CLDN18.2 Freund's Rec . Hu Alum gμ52 50 gμ Yes CLDN18.CpG20293HEK- Incomplete 2.5x106 cells 2.5x107 cells Yes CLDN18.2 Freund's 293HEK- Alum 112 2.5x106 cells 2.5x107 cells Yes CLDN18.CpG20 1293HEK- CLDN18.Incomplete 2.5x106 cells 2.5x107 cells Yes Freund's 293HEK- CLDN18.293HEK- CLDN18.

Alum 1.25x106 cells 1.25x107 cells No CpG20 1.25x106 cells 1.25x107 cells Incomplete Freund's Yes EpiMED Docket No .: SIBA064PCT Table 2. Stability of humanized anti - Claudin 18.2 antibodies at Time 0 and Time 1 ( upper panel ) , and Time 2 and Change ( lower panel ) . Time Antibody 5046ABSM 5046AFRS 5103FBSM 5103FFRS 5111DBSM 5194GBSM Date % POI % HMW % LMW Date Time % POI % HMW % LMW 9/10/21 93.98 0.86 5.16 9/30/21 94.29 0.5 5. 9/10/21 100 0 0 9/30/21 100 0 9/10/21 100 0 0 9/30/21 100 0 /22/21 92.48 7.52 0 11/11/21 93.74 6.26 9/10/21 100 0 0 9/30/21 100 0 12/1/21 99.54 0.46 0 12/20/21 99.44 0.56 5194GFRS 5194GGS /28/21 99.13 0 0 11/11/21 98.69 1.31 /28/21 97.2 3.18 0 11/11/21 99.86 0.14 Time Change Antibody Date % POI % HMW % LMW Weeks A % POI A % HMW A % LMW 5046ABSM 5046AFRS 5103FBSM 5103FFRS 5111DBSM 5194GBSM 5194GFRS 5194GGS /21/21 99.08 0.92 0 5.9 5.1 0.06 -5. /21/21 100 0 0 5.9 0 0 /21/21 100 0 0 5.9 0 0 11/30/21 93.32 6.68 0 5.6 0.84 -0.84 /21/21 100 0 0 5.9 0 1/6/22 99.26 0.74 0 5.1 -0.28 0. 11/30/21 97.82 2.18 0 4.7 -1.31 2.18 11/30/21 | 99.0.19 0 4.7 2.61 -2.99 21 Antibody Code EpiMED Docket No .: SIBA064PCT Table 3. Anti - CLDN18.2 antibodies and the levels of expression in CHO cells . Antibody Name Antibody Background ExpiCHO MFI ( 3ug / ml ) SI - 64M1 5103F3 BSM Humanized Rabbit 213 SI - 64CH5 5208H10 Human / Rabbit Chimeric 87 SI - 64C8 Zolbetuximab Human / Mouse Chimeric 107 SI - 64C- 72C1 LaNova Unknown 257 SI - 64C- AB011 – Carsgen Humanized Not available SI - 64C11 MIL93 - Mabworks Humanized Not available Table 4. OCTET affinity and avidity of anti - CLDN18.2 antibodies . OCTET Affinity OCTET Avidity KD ( M ) kon ( 1 / Ms ) kdiss ( 1 / s ) KD ( M ) kon ( 1 / Ms ) kdiss ( 1 / s ) SI - 64M1 1.45E - 09 5.23E + 05 7.61E - 04 2.16E - SI - 64CH5 3.00E - 09 9.50E + 04 2.85E - 3.79E + Not Tested 8.17E - SI - 64C8 1.59E - 09 2.18E + 05 3.46E - 04 5.31E - 09 5.81E + 05 3.08E - SI - 64C9 1.87E - 09 3.49E + 05 6.53E - 04 1.73E - 09 6.54E + 05 1.13E - SI - 64C10 4.35E - 10 4.74E + 05 2.06E - 04 4.56E - 10 5.38E + 05 2.45E - SI - 64C11 7.14E - 10 4.60E + 05 3.28E - 04 1.76E - 09 1.09E + 06 1.93E - Table 5. The binding specificity of humanized clones and controls to mouse and rat CLDN18.2 . Antibody Code Mouse Rat CLDN18.1 CLDN18.2 CLDN18.1 CLDN18.SI - 64M1 118 11759 119 87SI - 64CH5 89.1 210 126 26SI - 64C8 162 8024 144 55SI - 64C9 117 7729 262 51 22 EpiMED Docket No .: SIBA064PCT Table 6. Comparison of coding sequences for CDRs and framework of the variable region between 5103F3 BSM and other anti - CLDN18.2 antibodies . Zolbetuximab ( SI - 64C8 ) Variable Region CDR Only Framework Only | SI - 64C9 , SI - 64C10 , Average % Identity 73.87 68.6 75.SI - 64C( SD ) ( ± 6.7 ) ( ± 11.1 ) ( ± 6.0 ) 5103F3 BSM % Identity : 53.6 23.1 65.Two - Tailed , One- P - Value 0.0344 0.0192 0.09Sample T - Test P < 0.Significantly Different Yes Yes No 72C1 - LaNova ( SI - 64C9 ) SI - 64C8 , SI - 64C10 , Average % Identity 76.03 55.03 SI - 64C( SD ) ( ± 7.1 ) ( ± 7.5 ) ( ± 10.8 ) 5103F3 BSM % Identity : 56.68.Two - Tailed , One- P - Value 0.0397 0.0148 0.12Sample T - Test P < 0.Significantly Different Yes Yes No AB011 - Carsgen ( SI - 64C10 ) SI - 64C8 , SI - 64C9 , Average % Identity 68.5 53.63 74.SI - 64C( SD ) ( ± 1.5 ) ( ± 6.3 ) ( ± 4.44 ) 5103F3 BSM % Identity : 57.4 26.2 69.Two - Tailed , One- P - Value 0.0061 0.0171 0.21Sample T - Test P < 0.Significantly Different Yes Yes No MIL93 - Mabworks ( SI - 64C11 ) SI - 64C8 , SI - 64C9 , Average % Identity 77.6 60.93 84.SI - 64C( SD ) ( ± 7.7 ) ( ± 17.7 ) ( ± 10.7 ) 5103F3 BSM % Identity : 16.9 68.Two - Tailed , One- P - Value P < 0. 0.0334 0.0496 0.1Sample T - Test Significantly Different Yes Yes No 23 EpiMED Docket No .: SIBA064PCT SEQUENCE LISTING Antibody Variable Region Nucleic Acid Amino Acid SEQ ID NO . SEQ ID NO . Rabbit VH 5103FVL 5103F3 Humanized VH 5 BSM VL 7 5103F3 Chimeric VH 9 FRS VL 11 Rabbit VH 13 5208HVL 15 Rabbit VH 17 5046AVL 19 5046A2 Humanized VH 21 BSM VL 23 25046A2 Chimeric VH 25 FRS VL 27 5111D12 Rabbit VH 29 VL 31 5111DBSM Humanized VH 33 VL 35 5194G6 Rabbit VH 37 VL 39 5194GBSM Humanized VH 41 VL 43 5194G6 Chimeric VH 45 FRS VL 47 5194G6 GS VH 49 GS VL 51 5119H11 Rabbit VH 53 VL 55 Antibody VH VL CDRH1 CDRH2 CDRH3 CDRH1 CDRH2 CDRH5103F3 57 58 59 60 61 5208H10 63 64 65 66 67 5046A2 69 70 71 72 73 5111D12 75 76 77 78 79 5119H11 81 82 83 84 85 ﻼﻠﻫ EpiMED Docket No .: SIBA064PCT > Sequence ID 1 : 5103F3 Rabbit VH Nucleic Acid Sequence CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACAGCCCTGACACTCACCTGCA CAGTCTCTGGATTCTCCCTCAGTAGCTATTCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGG GCTGGAGTGGATCGGATATCTTAATACTGGTGGTAGCACATACTACGCGAGCTGGGTGAATGGT CGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCG AGGACACGGCCACCTATTTCTGTACCAGGGCCGGGGGTGTTAGTAGCAATATGGGCTTTAACAT CTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGC > Sequence ID 2 : 5103F3 Rabbit VH Amino Acid Sequence QSVEESGGRLVTPGTPLTLTCTVSGIDLSGFAMGWVRQAPGKGPEYIGFIDSGGGAFYATWARG RFTISRTSSTTVDLKMTSLTAADTATYFCARHGGNTYYYAMDPWGPGTLVTVSS > Sequence ID 3 : 5103F3 Rabbit VL Nucleic Acid Sequence GCCCTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGAGAGGCACAGTCACCATCA AGTGCCAGGCCAGTCAGAGCATTAGTAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCC TCCCAAGCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGC AGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTT ACTACTGTCAACAGGTGTATAGTGTCACTAATATTGATAATGCTTTCGGCGGAGGCACCGAGGT GGAGTTCAAA > Sequence ID 4 : 5103F3 Rabbit VL Amino Acid Sequence ALVMTQTPASVEAAVRGTVTIKCQASQSISNYLAWYQQKPGQPPKLLIYSASTLASGVSSRFKG SGSGTQFTLTISGVECADAATYYCQQVYSVTNIDNAFGGGTEVEFK > Sequence ID 5 : 5103F3 BSM VH Nucleic Acid Sequence CAGGTGCAGCTGCAGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGAGCCCCTGAGGCTGTCCT GCAAGACCTCCGGCATCGACCTGTCCGGCTTCGCCATGGGCTGGGTGAGGCAGGCCCCCGGCAA GGGCCTGGAGTACATCGGCTTCATCGACTCCGGCGGCGGCGCCTTCTACGCCACCTGGGCCAGG GGCAGGTTCACCATCTCCAGGACCTCCACCAACACCGTGTACCTGCAGATGAACTCCCTGACCG CCGAGGACACCGCCGTGTACTACTGCGCCAGGCACGGCGGCAACACCTACTACTACGCCATGGA CCCCTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 6 : 5103F3 BSM VH Amino Acid Sequence QVQLQESGGRLIKPGEPLRLSCKTSGIDLSGFAMGWVRQAPGKGLEYIGFIDSGGGAFYATWAR GRFTISRTSTNTVYLQMNSLTAEDTAVYYCARHGGNTYYYAMDPWGPGTLVTVSS > Sequence ID 7 : 5103F3 BSM VL Nucleic Acid Sequence GCCCTGAGGATGACCCAGTCCCCCTCCTCCCTGGCCGCCACCACCGGCCAGAGGGTGACCATCA CCTGCCAGGCCTCCCAGTCCATCTCCAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCC CCCCAAGCTGCTGATCTACTCCGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCAAGGGC EpiMED Docket No .: SIBA064PCT TCCGGCTCCGGCACCCAGTTCACCCTGACCATCTCCAGCGTGCAGCCCGAGGACTTCGCCACCT ACTACTGCCAGCAGGTGTACTCCGTGACCAACATCGACAACGCCTTCGGCGGCGGCACCAGGGT GGAGATCAAG > Sequence ID 8 : 5103F3 BSM VL Amino Acid Sequence ALRMTQSPSSLAATTGQRVTITCQASQSISNYLAWYQQKPGQPPKLLIYSASTLASGVPSRFKG SGSGTQFTLTISCVQCEDFATYYCQQVYSVTNIDNAFGGGTRVEIK > Sequence ID 9 : 5103F3 FRS VH Nucleic Acid Sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAGGCTGTCCT GCGCCGCCTCCGGCATCGACCTGTCCGGCTTCGCCATGGGCTGGGTGAGGCAGGCCCCCGGCAA GGGCCTGGAGTGGGTGGGCTTCATCGACTCCGGCGGCGGCGCCTTCTACGCCACCTGGGCCAGG GGCAGGTTCACCATCTCCAGGACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGG CCGAGGACACCGCCGTGTACTACTGCGCCAGGCACGGCGGCAACACCTACTACTACGCCATGGA CCCCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 10 : 5103F3 FRS VH Amino Acid Sequence QVQLVESGGGLVKPGGSLRLSCAASGIDLSGFAMGWVRQAPGKGLEWVGFIDSGGGAFYATWAR GRFTISRTSKNTVYLQMNSLRAEDTAVYYCARHGGNTYYYAMDPWGQGTLVTVSS > Sequence ID 11 : 5103F3 FRS VL Nucleic Acid Sequence GCCCTGGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCT CCTGCCAGGCCTCCCAGTCCATCTCCAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGC CCCCAAGCTGCTGATCTACTCCGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGC TCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCGTGGAGCCCGAGGACTTCGCCACCT ACTACTGCCAGCAGGTGTACTCCGTGACCAACATCGACAACGCCTTCGGCGGCGGCACCAAGGT GGAGATCAAG > Sequence ID 12 : 5103F3 FRS VL Amino Acid Sequence ALVMTQSPSSLSASVGDRVTISCQASQSISNYLAWYQQKPGQAPKLLIYSASTLASGVPSRFSG SGSGTDFTLTISSVEPEDFATYYCQQVYSVTNIDNAFGGGTKVEIK > Sequence ID 13 : 5208H10 Rabbit VH Nucleic Acid Sequence CAGTCGGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCGACGGATACCCTGACACTCACCTGCA CAGTCTCTGGATTCTCCCTCAGTACCTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAACGG GCTGGAATGGATCGGAATCATTGATGCTGTTGGTCGCACATATTACGCGAGTTGGGCGAAAAGT CGATCCACCATCACCAGGAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGTCTGACAG CCGCGGACACGGCCACCTATTTCTGTGCGAGATGGTGGGATCTCTGGGGCCAAGGCACCCTGGT CACCGTCTCGAGC > Sequence ID 14 : 5208H10 Rabbit VH Amino Acid Sequence 26 EpiMED Docket No .: SIBA064PCT QSVKESEGGLFKPTDTLTLTCTVSGFSLSTYAMSWVRQAPGNGLEWIGIIDAVGRTYYASWAKS RSTITRNTNLNTVTLKMTSLTAADTATYFCARWWDLWGQGTLVTVSS > Sequence ID 15 : 5208H10 Rabbit VL Nucleic Acid Sequence GCGCAAGTGCTGACCCAGACTGCATCGTCCGTGTCTGCCGCTGTGGGAGGCACTGTCACCATCA ATTGCCAGTCCAGTCAGAGTGTTTATGATAGCAACGACTTAACCTGGTATCAGCAGAAACCAGG GCAGTCTCCCAAGCTCCTGATCTATCTGGCATCCACTCTGGCATCTGGGGTCCCATCGCGATTC AAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATTCTG CCACTTACTACTGTGCAGCCCATTATAATAGTGATATTTATACTTTCGGCGGAGGGACCGAGGT GGAGGTCAAA > Sequence ID 16 : 5208H10 Rabbit VL Amino Acid Sequence AQVLTQTASSVSAAVGGTVTINCQSSQSVYDSNDLTWYQQKPGQSPKLLIYLASTLASGVPSRF KGSGSGTQFTLTISDLECDDSATYYCAAHYNSDIYTFGGGTEVEVK > Sequence ID 17 : 5046A2 Rabbit VH Nucleic Acid Sequence TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCAAGCTTA CGCTCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTG TCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGATAACGCCTGGAGGATCCCTGACACTCACCTGC ACCGTCTCTGGAATCGACCTCAGTAGCAATGGAATGAGCTGGGTCCGCCAGTCTCCAGGGAAGG GGCTGGAATACATCGGATTCATTGATACTGATGGTAGCGCATACTCCGCGAGCTGGGCGAAAGG CCGATTCACCATCTCCAAAGCCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACC GAGGACACGGCCACCTATTTCTGTGCCAGAGCCTATGTTAGTGGTAGTACTGGTTACAATTTTA ACATCTGGGGCCCGGGGACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGGCCCATCGGTCTT CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCT > Sequence ID 18 : 5046A2 Rabbit VH Amino Acid Sequence FSPQVSTPRSKFKRISSEFKLTLTMETGLRWLLLVAVLKGVQCQSVEESGGRLITPGGSLTLTC TVSGIDLSSNGMSWVRQSPGKGLEYIGFIDTDGSAYSASWAKGRFTISKASSTTVDLKMTSLTT EDTATYFCARAYVSGSTGYNFNIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC > Sequence ID 19 : 5046A2 Rabbit VL Nucleic Acid Sequence TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCAAGCTTC GAATCGACATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGG TGCCAGATGTGCATTCGAATTGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTGGGAGGCACA GTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGCCTGGTATCAGCAGAAAC CAGGGCAGCCTCCCAAGTCCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCCCATCGCG GTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGAT GCTGCCACTTACTACTGTCAACAGGGTTATACTATTCGTAATATTGATAATGCTTTCGGCGGAG GGACCGAGGTGGAGTTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA TGAGCAGTTGAAATCTGGAACTGCCTCT 27 EpiMED Docket No .: SIBA064PCT > Sequence ID 20 : 5046A2 Rabbit VL Amino Acid Sequence FSPQVSTPRSKFKRISSEFKLRIDMDTRAPTQLLGLLLLWLPGARCAFELTQTPSSVEAAVGGT VTIKCQASQSISSYLAWYQQKPGQPPKSLIYKASTLASGVPSRFKGSGSGTEFTLTISDLECAD AATYYCQQGYTIRNIDNAFGGGTEVEFKRTVAAPSVFIFPPSDEQLKSGTAS > Sequence ID 21 : 5046A2 BSM VH Nucleic Acid Sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGGCTCCCTGAGGCTGACCT GCAAGACCTCCGGCATCGACCTGTCCTCCAACGGCATGTCCTGGGTGAGGCAGCCCCCCGGCAA GGGCCTGGAGTACATCGGCTTCATCGACACCGACGGCTCCGCCTACTCCGCCTCCTGGGCCAAG GGCAGGTTCACCATCTCCAAGGCCTCCATCAACACCGTGTTCCTGCAGATGACCTCCCTGAAGT CCGAGGACACCGCCATCTACTACTGCGCCAGGGCCTACGTGTCCGGCTCCACCGGCTACAACTT CAACATCTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 22 : 5046A2 BSM VH Amino Acid Sequence QVQLVESGGRLIKPGGSLRLTCKTSGIDLSSNGMSWVRQPPGKGLEYIGFIDTDGSAYSASWAK GRFTISKASINTVFLQMTSLKSEDTAIYYCARAYVSGSTGYNFNIWGPGTLVTVSS > Sequence ID 23 : 5046A2 BSM VL Nucleic Acid Sequence GCCTTCAGGCTGACCCAGACCCCCTCCTCCTTCGCCGCCACCCTGGGCCAGAGGGTGACCATCA CCTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCAAGCC CCCCAAGTCCCTGATCTACAAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCAAGGGC TCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGACGACTTCGCCACCT ACTACTGCCAGCAGGGCTACACCATCAGGAACATCGACAACGCCTTCGGCGGCGGCACCAGGGT GGAGATCAAG > Sequence ID 24 : 5046A2 BSM VL Amino Acid Sequence AFRLTQTPSSFAATLGQRVTITCQASQSISSYLAWYQQKPGKPPKSLIYKASTLASGVPSRFKG SGSGTDFTLTISSLQPDDFATYYCQQGYTIRNIDNAFGGGTRVEIK > Sequence ID 25 : 5046A2 FRS VH Nucleic Acid Sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGATCAAGCCCGGCGGCTCCCTGAGGCTGTCCT GCGCCGCCTCCGGCATCGACCTGTCCTCCAACGGCATGTCCTGGGTGAGGCAGGCCCCCGGCAA GGGCCTGGAGTGGGTGGGCTTCATCGACACCGACGGCTCCGCCTACTCCGCCTCCTGGGCCAAG GGCAGGTTCACCATCTCCAAGGACTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGG CCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACGTGTCCGGCTCCACCGGCTACAACTT CAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 26 : 5046A2 FRS VH Amino Acid Sequence QVQLVESGGGLIKPGGSLRLSCAASGIDLSSNGMSWVRQAPGKGLEWVGFIDTDGSAYSASWAK GRFTISKDSKNTVYLQMNSLRAEDTAVYYCARAYVSGSTGYNFNIWGQGTLVTVSS 28 EpiMED Docket No .: SIBA064PCT > Sequence ID 27 : 5046A2 FRS VL Nucleic Acid Sequence GCCTTCGTGCTGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCT CCTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGC CCCCAAGTCCCTGATCTACAAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGC TCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGGAGCCCGAGGACTTCGCCACCT ACTACTGCCAGCAGGGCTACACCATCAGGAACATCGACAACGCCTTCGGCGGCGGCACCAAGGT GGAGATCAAG > Sequence ID 28 : 5046A2 FRS VL Amino Acid Sequence AFVLTQSPSSLSASVGDRVTISCQASQSISSYLAWYQQKPGQAPKSLIYKASTLASGVPSRFSG SGSGTDFTLTISSLEPEDFATYYCQQGYTIRNIDNAFGGGTKVEIK > Sequence ID 29 : 5111D12 Rabbit VH Nucleic Acid Sequence TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCAAGCTTA CGCTCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGNNCGCTGTGCTCAAAGGTGTCCAGTG TCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACAGCCCTGACACTCACCTGC ACAGTCTCTGGATTCTCCCTCAGTAGCTATTCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGG GGCTGGAGTGGATCGGATATCTTAATACTGGTGGTAGCACATACTACGCGAGCTGGGTGAATGG TCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACC GAGGACACGGCCACCTATTTCTGTACCAGGGCCGGGGGTGTTAGTAGCAATATGGGCTTTAACA TCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGNNCCATCGGTCTTCCC CCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC > Sequence ID 30 : 5111D12 Rabbit VH Amino Acid Sequence FSPQVSTPRSKFKRISSEFKLTLTMETGLRWLLLXAVLKGVQCQSVEESGGRLVTPGTALTLTC TVSGFSLSSYSMGWVRQAPGKGLEWIGYLNTGGSTYYASWVNGRFTISKTSSTTVDLKITSPTT EDTATYFCTRAGGVSSNMGFNIWGQGTLVTVSSASTKXPSVFPLAPSSKSTSGGTAALG > Sequence ID 31 : 5111D12 Rabbit VL Nucleic Acid Sequence TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCAAGCTTC GAATCGACATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGG TGCCAGATGTGACCCTGTGCTGACCCAGACTCCAGCCTCCGTGTCTGAGCCTGTGGGAGGCACA GTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGCCTGGTATCAGCAGAAAC CAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCCCATCGCG GTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGAT GCTGCCACTTACTACTGTCAATGTACTGATTATGGCTACAATTATCTTGGGGCTTTCGGCGGAG GGACCGAGGTGGAGTTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA TGAGCAGTTGAAATCTGGAACTGCCTCTG > Sequence ID 32 : 5111D12 Rabbit VL Amino Acid Sequence 29 EpiMED Docket No .: SIBA064PCT FSPQVSTPRSKFKRISSEFKLRIDMDTRAPTQLLGLLLLWLPGARCDPVLTQTPASVSEPVGGT VTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTQFTLTISDLECAD AATYYCQCTDYGYNYLGAFGGGTEVEFKRTVAAPSVFIFPPSDEQLKSGTAS > Sequence ID 33 : 5111D12 BSM VH Nucleic Acid Sequence CAGGTGCAGCTGCTGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGAGTCCCTGAAGCTGACCT GCAAGACCTCCGGCTTCTCCCTGTCCTCCTACTCCATGGGCTGGGTGAGGCAGGCCCCCGGCAA GGGCCTGGAGTGGATCGGCTACCTGAACACCGGCGGCTCCACCTACTACGCCTCCTGGGTGAAC GGCAGGTTCACCATCTCCAAGACCTCCATCTCCACCGTGTTCCTGCAGCTGAGGTCCCCCAGGT CCGAGGACACCGCCCTGTACTACTGCACCAGGGCCGGCGGCGTGTCCTCCAACATGGGCTTCAA CATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 34 : 5111D12 BSM VH Amino Acid Sequence QVQLLESGGRLIKPGESLKLTCKTSGFSLSSYSMGWVRQAPGKGLEWIGYLNTGGSTYYASWVN GRFTISKTSISTVFLQLRSPRSEDTALYYCTRAGGVSSNMGFNIWGQGTLVTVSS > Sequence ID 35 : 5111D12 BSM VL Nucleic Acid Sequence GACCCCGTGCTGACCCAGTCCCCCTCCTTCCTGTCCCTGACCGTGGGCCAGAGGGTGACCATCA ACTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCC CCCCAAGCTGCTGATCTACAGGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGC TCCGGCTCCGGCACCGAGTTCACCCTGACCATCTCCTCCCTGCAGTCCGACGACTTCGCCACCT ACTACTGCCAGTGCACCGACTACGGCTACAACTACCTGGGCGCCTTCGGCGGCGGCACCAGGGT GGAGATCAAG > Sequence ID 36 : 5111D12 BSM VL Amino Acid Sequence DPVLTQSPSFLSLTVGQRVTINCQASQSISSYLAWYQQKPGQPPKLLIYRASTLASGVPSRFSG SGSGTEFTLTISSLQSDDFATYYCQCTDYGYNYLGAFGGGTRVEIK > Sequence ID 37 : 5194G6 Rabbit VH Nucleic Acid Sequence CAGGAGCAGCTGAAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGGGGGATCCCTGACACTCACCT GCAAAGCCTCTGGATTCTCCTTCAGTAGCGTCTACTACATGTGCTGGGTCCGCCAGGCTCCAGG GAAGGGGCTGGAGTGGATCGGATGCAGTTATACGAGTGATGGTAGCGCTTACTACGCGAACTGG GCGACAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACGAGTC TGACAGACGCGGACACGGCCACCTATTTCTGTGCGAGAGATAGGACTGATAGTCGTGATTATTT TTTTGACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCGAGC > Sequence ID 38 : 5194G6 Rabbit VH Amino Acid Sequence QEQLKESGGGLVQPGGSLTLTCKASGFSFSSVYYMCWVRQAPGKGLEWIGCSYTSDGSAYYANW ATGRFTISKTSSTTVTLQMTSLTDADTATYFCARDRTDSRDYFFDLWGPGTLVTVSS > Sequence ID 39 : 5194G6 Rabbit VL Nucleic Acid Sequence EpiMED Docket No .: SIBA064PCT GCCTATGATATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCAGCATCA GTTGTCAGTCCAGTCAGAATGTTTATAGTAACTACTTATCCTGGTATCAGCAGAAACCAGGGCA GCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCTTCTGGGGTCTCATCGCGATTCAGA GGCAGTGGATCCGGGACACAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCA CTTACTATTGTCAACAGGGTTATAAATATGGTAATGTTAATAATCTTTTCGGCGGAGGGACCGA GGTGGAGGTCAAA > Sequence ID 40 : 5194G6 Rabbit VL Amino Acid Sequence AYDMTQTPASVEAAVGGTVSISCQSSQNVYSNYLSWYQQKPGQPPKLLIYRASTLASGVSSRFR GSGSGTQFTLTISGVECADAATYYCQQGYKYGNVNNLFGGGTEVEVK > Sequence ID 41 : 5194G6 BSM VH Nucleic Acid Sequence GAGGAGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCT GCACCGCCTCCGGCTTCACCTTCGAGTCCGTGTACTACATGTGCTGGGTGAGGCAGGCCCCCGG CAAGGGCCTGGAGTGGATCGGCTGCTCCTACACCTCCGACGGCTCCGCCTACTACGCCAACTGG GCCACCGGCAGGTTCACCATCTCCAAGACCTCCATCAACACCGTGTTCCTGCAGATGAGGTCCC TGAGGTCCGAGGACACCGCCATCTACTACTGCGCCAGGGACAGGACCGACTCCAGGGACTACTT CTTCGACCTGTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 42 : 5194G6 BSM VH Amino Acid Sequence EEQLLESGGGLVQPGGSLRLSCTASGFTFESVYYMCWVRQAPGKGLEWIGCSYTSDGSAYYANW ATGRFTISKTSINTVFLQMRSLRSEDTAIYYCARDRTDSRDYFFDLWGPGTLVTVSS > Sequence ID 43 : 5194G6 BSM VL Nucleic Acid Sequence GCCTACAGGATGACCCAGTCCCCCTCCTCCTTCTCCGCCTCCACCGGCCAGAGGGTGACCATCA CCTGCCAGTCCTCCCAGAACGTGTACTCCAACTACCTGTCCTGGTACCAGCAGAAGCCCGGCAA GCCCCCCAAGCTGCTGATCTACAGGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCAGG GGCTCCGGCTCCGGCACCCAGTTCACCCTGACCATCTCCTGCGTGCAGTGCGACGACTTCGCCA CCTACTACTGCCAGCAGGGCTACAAGTACGGCAACGTGAACAACCTGTTCGGCGGCGGCACCAG GGTGGAGATCAAG > Sequence ID 44 : 5194G6 BSM VL Amino Acid Sequence AYRMTQSPSSFSASTGQRVTITCQSSQNVYSNYLSWYQQKPGKPPKLLIYRASTLASGVPSRFR GSGSGTQFTLTISCVQCDDFATYYCQQGYKYGNVNNLFGGGTRVEIK > Sequence ID 45 : 5194G6 FRS VH Nucleic Acid Sequence CAGGAGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCT GCGCCGCCTCCGGCTTCACCTTCTCCTCCGTGTACTACATGTGCTGGGTGAGGCAGGCCCCCGG CAAGGGCCTGGAGTGGGTGGGCTGCTCCTACACCTCCGACGGCTCCGCCTACTACGCCAACTGG GCCACCGGCAGGTTCACCATCTCCAAGACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCC 31 EpiMED Docket No .: SIBA064PCT TGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACAGGACCGACTCCAGGGACTACTT CTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 46 : 5194G6 FRS VH Amino Acid Sequence QEQLVESGGGLVQPGGSLRLSCAASGFTFSSVYYMCWVRQAPGKGLEWVGCSYTSDGSAYYANW ATGRFTISKTSKNTVYLQMNSLRAEDTAVYYCARDRTDSRDYFFDLWGQGTLVTVSS > Sequence ID 47 : 5194G6 FRS VL Nucleic Acid Sequence GCCTACGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCT CCTGCCAGTCCTCCCAGAACGTGTACTCCAACTACCTGTCCTGGTACCAGCAGAAGCCCGGCCA GGCCCCCAAGCTGCTGATCTACAGGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCC GGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCGTGGAGCCCGAGGACTTCGCCA CCTACTACTGCCAGCAGGGCTACAAGTACGGCAACGTGAACAACCTGTTCGGCGGCGGCACCAA GGTGGAGATCAAG > Sequence ID 48 : 5194G6 FRS VL Amino Acid Sequence AYVMTQSPSSLSASVGDRVTISCQSSQNVYSNYLSWYQQKPGQAPKLLIYRASTLASGVPSRFS GSGSGTDFTLTISSVEPEDFATYYCQQGYKYGNVNNLFGGGTKVEIK > Sequence ID 49 : 5194G6 GS VH Nucleic Acid Sequence CAGGAGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCT GCGCCGCCTCCGGCTTCACCTTCTCCTCCGTGTACTACATGTGCTGGGTGAGGCAGGCCCCCGG CAAGGGCCTGGAGTGGGTGGGCTGCTCCTACACCTCCGACGGCTCCGCCTACTACGCCAACTGG GCCACCGGCAGGTTCACCATCTCCAAGACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCC TGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACAGGACCGACTCCAGGGACTACTT CTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 50 : 5194G6 GS VH Amino Acid Sequence QEQLLESGGGLVQPGGSLRLSCAASGFTFSSVYYMCWVRQAPGKGLEWVGCSYTSDGSAYYANW ATGRFTISKTSKNTVYLQMNSLRAEDTAVYYCARDRTDSRDYFFDLWGQGTLVTVSS > Sequence ID 51 : 5194G6 GS VL Nucleic Acid Sequence GCCTACAGGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCT CCTGCCAGTCCTCCCAGAACGTGTACTCCAACTACCTGTCCTGGTACCAGCAGAAGCCCGGCCA GGCCCCCAAGCTGCTGATCTACAGGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCC GGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCGGCGTGCAGTCCGAGGACTTCGCCA CCTACTACTGCCAGCAGGGCTACAAGTACGGCAACGTGAACAACCTGTTCGGCGGCGGCACCAA GGTGGAGATCAAG > Sequence ID 52 : 5194G6 GS VL Amino Acid Sequence 32 EpiMED Docket No .: SIBA064PCT AYRMTQSPSSVSASVGDRVTISCQSSQNVYSNYLSWYQQKPGQAPKLLIYRASTLASGVPSRFS GSGSGTDFTLTISGVQSEDFATYYCQQGYKYGNVNNLFGGGTKVEIK > Sequence ID 53 : 5119H11 Rabbit VH Nucleic Acid Sequence CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCA CGGTCTCTGGATTCTCCCTCAGTACCTATTCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGG GCTGGAATGGATCGGAGTCATTTACTATGGTGAGCCCACATACTGCGCGGCCTGGGCGAAAGGC CGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCG AGGACACGGCCACCTATTTCTGTGCCAGATCAGGTGATCGTAATGGCTACTACTTTAACATCTG GGGCCCAGGCACCCTGGTCACCGTCTCGAGC > Sequence ID 54 : 5119H11 Rabbit VH Amino Acid Sequence QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYSMGWVRQAPGKGLEWIGVIYYGEPTYCAAWAKG RFTISKTSSTTVDLKITSPTTEDTATYFCARSGDRNGYYFNIWGPGTLVTVSS > Sequence ID 55 : 5119H11 Rabbit VL Nucleic Acid Sequence GCCTATGATATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCA AGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCC TCCCAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGC AGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGCCGATGCAGCCACTT ACTACTGTCAACAGGTTGCTGTTATTGGTAATGTTGGGGATAATACTTTCGGCGGAGGGACCGA GGTGGAGGTCAAA > Sequence ID 56 : 5119H11 Rabbit VL Amino Acid Sequence AYDMTQTPASVSAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYYASTLASGVPSRFKG SGSGTEFTLTISGVQCADAATYYCQQVAVIGNVGDNTFGGGTEVEVK > Sequence ID 57 : 5103F3 VH - CDRHGFAMG > Sequence ID 58 : 5103F3 VH - CDRHFIDSGGGAFYATWARG > Sequence ID 59 : 5103F3 VH - CDRHHGGNTYYYAMDP > Sequence ID 60 : 5103F3 VL - CDRLQASQSISNYLA > Sequence ID 61 : 5103F3 VL - CDRL 33 EpiMED Docket No .: SIBA064PCT SASTLAS > Sequence ID 62 : 5103F3 VL - CDRLQQVYSVTNIDNA > Sequence ID 63 : 5208H10 VH - CDRHTYAMS > Sequence ID 64 : 5208H10 VH - CDRHIIDAVGRTYYASWAKS > Sequence ID 65 : 5208H10 VH - CDRHWWDL > Sequence ID 66 : 5208H10 VL - CDRLQSSQSVYDSNDLT > Sequence ID 67 : 5208H10 VL - CDRLLASTLASGVPS > Sequence ID 68 : 5208H10 VL - CDRLAAHYNSDIYT > Sequence ID 69 : 5046A2 VH - CDRHSNGMS > Sequence ID 70 : 5046A2 VH - CDRHFIDTDGSAYSASWAKG > Sequence ID 71 : 5046A2 VH - CDRHAYVSGSTGYNFNI > Sequence ID 72 : 5046A2 VL - CDRLQASQSISSYLA > Sequence ID 73 : 5046A2 VL - CDRLKASTLAS > Sequence ID 74 : 5046A2 VL - CDRLQQGYTIRNIDNA 34 EpiMED Docket No .: SIBA064PCT > Sequence ID 75 : 5111D12 VH - CDRHSYSMG > Sequence ID 76 : 5111D12 VH - CDRHYLNTGGSTYYASWVNG > Sequence ID 77 : 5111D12 VH - CDRHAGGVSSNMGFNI > Sequence ID 78 : 5111D12 VL - CDRLQASQSISSYLA > Sequence ID 79 : 5111D12 VL - CDRLRASTLAS > Sequence ID 80 : 5111D12 VL - CDRLQCTDYGYNYLGA > Sequence ID 81 : 5119H11 VH - CDRHTYSMG > Sequence ID 82 : 5119H11 VH - CDRHVIYYGEPTYCAAWAKG > Sequence ID 83 : 5119H11 VH - CDRHSGDRNGYYFNI > Sequence ID 84 : 5119H11 VL - CDRLQASQSISSYLA > Sequence ID 85 : 5119H11 VL - CDRLYASTLAS > Sequence ID 86 : 5119H11 VL - CDRLQQVAVIGNVGDNT > Sequence ID 87 : Human Claudin18.2 Nucleic Acid Sequence ATGGCCGTGACCGCCTGCCAGGGCCTGGGCTTCGTGGTGAGCCTGATCGGCATCGCCGGCATCA TCGCCGCCACCTGCATGGACCAGTGGAGCACCCAGGACCTGTACAACAACCCCGTGACCGCCGT GTTCAACTACCAGGGCCTGTGGAGGAGCTGCGTGAGGGAGAGCAGCGGCTTCACCGAGTGCAGG GGCTACTTCACCCTGCTGGGCCTGCCCGCCATGCTGCAGGCCGTGAGGGCCCTGATGATCGTGG EpiMED Docket No .: SIBA064PCT GCATCGTGCTGGGCGCCATCGGCCTGCTGGTGAGCATCTTCGCCCTGAAGTGCATCAGGATCGG CAGCATGGAGGACAGCGCCAAGGCCAACATGACCCTGACCAGCGGCATCATGTTCATCGTGAGC GGCCTGTGCGCCATCGCCGGCGTGAGCGTGTTCGCCAACATGCTGGTGACCAACTTCTGGATGA GCACCGCCAACATGTACACCGGCATGGGCGGCATGGTGCAGACCGTGCAGACCAGGTACACCTT CGGCGCCGCCCTGTTCGTGGGCTGGGTGGCCGGCGGCCTGACCCTGATCGGCGGCGTGATGATG TGCATCGCCTGCAGGGGCCTGGCCCCCGAGGAGACCAACTACAAGGCCGTGAGCTACCACGCCA GCGGCCACAGCGTGGCCTACAAGCCCGGCGGCTTCAAGGCCAGCACCGGCTTCGGCAGCAACAC CAAGAACAAGAAGATCTACGACGGCGGCGCCAGGACCGAGGACGAGGTGCAGAGCTACCCCAGC AAGCACGACTACGTGTGA > Sequence ID 88 : Human Claudin18.1 Nucleic Acid Sequence ATGTCCACCACCACATGCCAAGTGGTGGCGTTCCTCCTGTCCATCCTGGGGCTGGCCGGCTGCA TCGCGGCCACCGGGATGGACATGTGGAGCACCCAGGACCTGTACGACAACCCCGTCACCTCCGT GTTCCAGTACGAAGGGCTCTGGAGGAGCTGCGTGAGGCAGAGTTCAGGCTTCACCGAATGCAGG CCCTATTTCACCATCCTGGGACTTCCAGCCATGCTGCAGGCAGTGCGAGCCCTGATGATCGTAG GCATCGTCCTGGGTGCCATTGGCCTCCTGGTATCCATCTTTGCCCTGAAATGCATCCGCATTGG CAGCATGGAGGACTCTGCCAAAGCCAACATGACACTGACCTCCGGGATCATGTTCATTGTCTCA GGTCTTTGTGCAATTGCTGGAGTGTCTGTGTTTGCCAACATGCTGGTGACTAACTTCTGGATGT CCACAGCTAACATGTACACCGGCATGGGTGGGATGGTGCAGACTGTTCAGACCAGGTACACATT TGGTGCGGCTCTGTTCGTGGGCTGGGTCGCTGGAGGCCTCACACTAATTGGGGGTGTGATGATG TGCATCGCCTGCCGGGGCCTGGCACCAGAAGAAACCAACTACAAAGCCGTTTCTTATCATGCCT CAGGCCACAGTGTTGCCTACAAGCCTGGAGGCTTCAAGGCCAGCACTGGCTTTGGGTCCAACAC CAAAAACAAGAAGATATACGATGGAGGTGCCCGCACAGAGGACGAGGTACAATCTTATCCTTCC AAGCACGACTATGTGTAA 36

Claims (30)

EpiMED Docket No .: SIBA064PCT ANTI - CLAUDIN18.2 ANTIBODY AND METHOD OF MAKING AND USING THEREOF What is claimed is : CLAIMS

1. An isolated monoclonal antibody ( mAb ) or antigen - binding fragment thereof having a binding specificity to CLDN18 , comprising an amino acid sequence having at least 90 % identity with the SEQ ID NO : 2 , 4 , 6 , 8 , 10 , 12 , 14 , 16 , 18 , 20 , 22 , 24 , 26 , 28 , 30 , 32 , 34 , 36 , 38 , 40 , 42 , , 46 , 48 , 50 , 52 , 54 , or 56 .

2. The isolated mAb or antigen - binding fragment of Claim 1 , wherein the isolated mAb or antigen - binding fragment thereof selectively binds to CLDN18.2 but not CLDN18.1 .

3. The isolated mAb or antigen - binding fragment thereof of Claim 1 , further comprising a human framework region .

4. The isolated mAb or antigen - binding fragment thereof of Claim 1 , wherein the isolated mAb is a humanized antibody , a chimeric antibody , or a recombinant antibody .

5. The isolated mAb or antigen - binding fragment thereof of Claim 1 , wherein the isolated mAb is an IgG .

6. The isolated mAb or antigen - binding fragment thereof of Claim 1 , wherein the antigen - binding fragment is a Fv , a Fab , a F ( ab ' ) 2 , a scFv , or a scFv2 fragment .

7. The isolated mAb or antigen - binding fragment thereof of Claim 1 , wherein the isolated mAb is a bispecific antibody , tri - specific antibody , or multi - specific antibody .

8. The isolated mAb or antigen - binding fragment thereof of Claim 1 , comprising an IgGheavy chain having an amino acid sequence having at least 90 % identity with the SEQ ID NO : 2 , , 10 , 14 , 18 , 22 , 26 , 30 , 34 , 38 , 42 , 46 , 50 , or 54 .

9. The isolated mAb or antigen - binding fragment thereof of Claim 1 , comprising a kappa light chain having an amino acid sequence having at least 90 % identity the SEQ ID NO : 4 , 8 , 12 , , 20 , 24 , 28 , 32 , 36 , 40 , 44 , 48 , 52 , or 56 .

10. The isolated mAb or antigen - binding fragment thereof of Claim 1 , comprising a variable heavy chain having an amino acid sequence having at least 90 % identity with the SEQ ID NO : 2 , 6 , 10 , 14 , 18 , 22 , 26 , 30 , 34 , 38 , 42 , 46 , 50 , or 54 , or a variable light chain having an amino acid sequence having at least 90 % identity with the SEQ ID NO : 4 , 8 , 12 , 16 , 20 , 24 , 28 , , 36 , 40 , 44 , 48 , 52 , or 56 .

11. An isolated mAb or antigen - binding fragment thereof having a binding specificity to CLDN18 , wherein the isolated mAb or antigen - binding fragment thereof comprises a variable heavy chain having complementarity determining region ( CDR ) 1 , CDR2 , and CDR3 of SEQ ID NO : 57 , 58 , and 59 ; 63 , 64 , and 65 ; 69 , 70 , and 71 ; 75 , 76 , and 77 ; or 81 , 82 , and 83 ; and a variable light chain having CDR 1 , CDR2 , and CDR3 of SEQ ID NO : 60 , 61 , and 62 ; 66 , 67 , and ; 72 , 73 , and 74 ; 78 , 79 , and 80 ; or 84 , 85 , and 86 . 37 EpiMED Docket No .: SIBA064PCT

12. An isolated nucleic acid encoding the isolated mAb or antigen - binding fragment of Claim 1 , the IgG1 heavy Chain of Claim 8 , the kappa light chain of Claim 9 , the variable light chain of Claim 10 , or the variable heavy chain of Claim 11 .

13. An expression vector comprising the isolated nucleic acid of Claim 12 , wherein the vector is expressible in a cell .

14. A host cell comprising the nucleic acid of Claim 12 , wherein the host cell is a prokaryotic cell or a eukaryotic cell .

15. A method of producing an antibody comprising culturing the host cell of one of Claim 14 , so that the antibody is produced .

16. An immuno - conjugate , comprising the isolated mAb or antigen - binding fragment thereof of Claim 1 conjugated to a drug unit through a linker , wherein the linker comprises a covalent bond selected from an ester bond , an ether bond , an amine bond , an amide bond , a disulfide bond , an imide bond , a sulfone bond , a phosphate bond , a phosphorus ester bond , a peptide bond , a hydrazone bond or a combination thereof .

17. The immuno - conjugate according to claim 16 , wherein the drug unit comprises a cytotoxic agent , an immune regulatory reagent , or a combination thereof .

18. The immuno - conjugate according to claim 16 , wherein the cytotoxic agent is selected from a growth inhibitory agent or a chemotherapeutic agent from a class of tubulin binders , DNA intercalators , DNA alkylators , enzyme inhibitors , immune modulators , antimetabolite agents , radioactive isotopes , or a combination thereof .

19. The immuno - conjugate according to claim 16 , wherein the cytotoxic agent is selected from a calicheamicin , ozogamicin , monomethyl auristatin E , emtansinea derivative or a combination thereof .

20. The immuno - conjugate according to claim 16 , wherein the immune regulatory reagents activate or suppress immune cells , T cell , NK cell , B cell , macrophage , or dendritic cell .

21. A pharmaceutical composition , comprising the isolated mAb or antigen - binding fragment thereof of Claim 1 or the immuno - conjugate of Claim 19 , and a pharmaceutically acceptable carrier .

22. The pharmaceutical composition of Claim 21 , further comprising a radioisotope , a radionuclide , a toxin , a therapeutic agent , a chemotherapeutic agent or a combination thereof .

23. The pharmaceutical composition of Claim 22 , wherein the therapeutic agent comprises an anti - estrogen agent , a receptor tyrosine kinase inhibitor , a kinase inhibitor , a cell cycle inhibitor , a DNA , RNA or protein synthesis inhibitor , a RAS inhibitor , or a combination thereof .

24. A method of treating a subject with a cancer , comprising administering to the subject an effective amount of the isolated mAb or antigen - binding fragment thereof of Claim 1 .

25. The method of Claim 24 , wherein the cancer comprises cells expressing Claudin 18.2 .

26. The method of Claim 24 , wherein the cancer comprises colorectal cancer , pancreatic cancer , esophageal cancer , nasopharyngeal cancer , anal cancer , rectal cancer , gastric cancer , or bladder cancer . 38 EpiMED Docket No .: SIBA064PCT

27. The method of Claim 24 , further comprising co - administering an effective amount of a therapeutic agent .

28. The method of Claim 24 , wherein the therapeutic agent comprises an antibody , a chemotherapy agent , an enzyme , an anti - estrogen agent , a receptor tyrosine kinase inhibitor , a kinase inhibitor , a cell cycle inhibitor , a DNA , RNA or protein synthesis inhibitor , a RAS inhibitor or a combination thereof .

29. The method of Claim 24 , wherein the subject is a human .

30. A solution comprising an effective concentration of the isolated mAb or an antigen- binding fragment thereof of Claim 1 , wherein the solution is blood plasma in a subject . 39