VHH polypeptides that bind to claudin-18, compositions and methods of use thereof

Abstract

Single domain VHH polypeptides (antibodies) that bind Claudin-18 (e.g., Claudin-18.2), VHH polypeptide products, methods, cells, pharmaceutical compositions, and kits. The VHH polypeptides may be recombinantly produced and expressed. Provided are also chimeric antigen receptor (CAR) polypeptides comprising the VHH polypeptides and CAR immune effector cells comprising the expressing the same.

Description

[0001] VHH POLYPEPTIDES THAT BIND TO CLAUDIN-18, COMPOSITIONS AND METHO DS O F USE THEREO F CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims priority to and the benefit of U. S. Provisional Application No.

[0003] 63 / 703,041, filed October 3, 2024, the entire contents of which are incorporated herein by reference.

[0004] BACKGROUND

[0005] Claudin isoform variant 18.2 (CLDN18.2) is a tight junction protein that has been identified in many different neoplasias, including gastric cancers, gastroesophageal adenocarcinomas, pancreatic carcinomas, and lung adenocarcinomas. Chimeric antigen receptor (CAR) T-cell therapy is one method for treating a neoplasia associated with CLDN18.2. However, to develop effective CAR-T-cell therapies, it is necessary to identify antigen-binding domains suitable for use in targeting the CAR proteins expressed by the CAR-T-cells to neoplasia cells surface-expressing CLDN18.2.

[0006] Accordingly, there is a need for improved antigen binding domains capable of binding a CLDN18.2 antigen.

[0007] SUMMARY

[0008] As described below, the disclosure provides single domain VHH polypeptides (nanobodies) that bind CLDN18 (e.g., CLDN18.2), VHH polypeptide products, methods of use, cells, pharmaceutical compositions, and kits. The V HH polypeptides may be recombinantly produced and expressed. Provided are also chimeric antigen receptor (CAR) polypeptides comprising the V HH polypeptides and CAR immune effector cells comprising and expressing the same.

[0009] In one aspect, the disclosure features a V H-heavy chain only ( V HH) polypeptide or an antigen binding portion thereof that specifically binds to a Claudin-18.1 (CLDN18.1) and / or Claudin- 18.2 (CLDN18.2) polypeptide or a fragment thereof. The V HH polypeptide or the antigen binding portion thereof contains three Complementarity Determining Regions (CDRs): CDR1, CDR2 and CDR3, which are structurally positioned between four camelid VHH framework ( FR) regions ( FR1-FR4) as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The three CDRs are selected from: CDR1 containing the amino acid sequence GRTF S SYA; CDR2 containing the amino acid sequence I S W SGGKT; and CDR3 containing an amino acid sequence selected from one or more of AAERDL L W LRYWDY; VAERDL L W LRYWDY; AAERDLRW LRYWDY; AAERDL L W LRYWDD;

[0010] AAERDL L L LRYWDY; AAERDL L W LRDWDY; AAERDRL W LRYWDY;

[0011] AAERN L L W LRYWDY; ATERDL L W LRYWDY; and AGERDL L W LRYWDY; CDR1 containing the amino acid sequence GRTF S SYI; CDR2 containing the amino acid sequence I S W SGDST; and CDR3 containing the amino acid sequence AAGRTTWANYAMDY; CDR1 containing the amino acid sequence GRTF S SYI; CDR2 containing the amino acid sequence I S W SGGST; and CDR3 containing the amino acid sequence AAGSTTWATYS MDY; CDR1 containing the amino acid sequence GRTL S S FA; CDR2 containing the amino acid sequence I S W SGGIT; and CDR3 containing the amino acid sequence AAGSTTWATYS MDY.

[0012] In another aspect, the disclosure features an antibody-drug conjugate containing a VHH polypeptide of any aspect of the disclosure, or embodiments thereof.

[0013] In another aspect, the disclosure features a polypeptide that specifically binds to Claudin-18.1 (CLDN18.1) or Claudin-18.2 (CLDN18.2), where the polypeptide or a CLDN18.1 and / or CLDN18.2-binding portion thereof has at least 85% amino acid sequence identity to a sequence selected from one or more of:

[0014] VQL VESGGGL VQAGGS LRL SCAASGRTF S SYAMGW FRQAPGKEREF VAAIS W SGGKTYYADS LKGRFTISRDNAKNTVYLQMN S LKPEDTAVYYCAAERDL L W LRYWDYWGQGTS VTV S S PTP APTI;

[0015] VQL VESGGGL VQAGGS LRL SCAASGRTF S SYIMGW FRQAPGKEREWAGIS W SGDSTYYADS VKGRFTISRDNGKNTVYLQMN S LKPEDTAVYYCAAGRTTWANYAMDYWGQGTS VTV S S PTP APTI;

[0016] VQL VESGGGL VQAGGS LRL SCAASGRTF S SYIMGW FRQAPGKEREF VAAIS W SGGSTYYDDS VKGRFTISRDNAKNTVYLQMN S LKPEDTAVYYCAAGSTTWATYSMDYWGQGTS VTV S S PTP APTI; and VQL VESGGGLAQAGGS LRL SCAASGRTL S S FAMGW FRQAPGKEREF VAAIS W SGGITYYADS VKGRFTISRDNAKNTVYLQMN S LKTEDTAVYYCAAGSTTWATYSMDYWGQGTS VTV S S PTP APTI.

[0017] In another aspect, the disclosure features a VH-heavy chain only (VHH) polypeptide or an antigen binding portion thereof that specifically binds to CLDN18.1 and / or CLDN18.2, where the binding protein or the antigen binding portion thereof contains an amino acid sequence with at least about 95% identity to one of the following sequences: VQL VESGGGL VQAGGS LRL SCAASGRTF S SYAMGW FRQAPGKEREF VAAIS W SGGKTYYADS LKGRFTISRDNAKNTVYLQM N S LKPEDTAVYYCAAERDL L W LRYV VDYWGQGTS VTV S S PTP APTI;

[0018] VQL VESGGGL VQAGGS LRL SCAASGRTF S SYIMGW FRQAPGKEREV VAGIS W SGDSTYYADS VKGRFTISRDNGKNTVYLQM N S LKPEDTAVYYCAAGRTTV VANYAMDYWGQGTS VTV S S PTP APTI;

[0019] VQL VESGGGL VQAGGS LRL SCAASGRTF S SYIMGW FRQAPGKEREF VAAIS W SGGSTYYDDS VKGRFTISRDNAKNTVYLQM N S LKPEDTAVYYCAAGSTTV VATYS MDYWGQGTS VTV S S PTP APTI; and VQL VESGGGLAQAGGS LRL SCAASGRTL S S FAMGW FRQAPGKEREF VAAI S W SGGITYYADS VKGRFTI SRDNAKNTVYLQM N S LKTEDTAVYYCAAGSTTWATYS MDYWGQGTS VTV S S PTP APTI.

[0020] In another aspect, the disclosure features a polynucleotide encoding the VH-heavy chain only (VHH) polypeptide of any aspect of the disclosure or embodiments thereof.

[0021] In another aspect, the disclosure features a vector containing the polynucleotide of any aspect of the disclosure or embodiments thereof.

[0022] In another aspect, the disclosure features a VH-heavy chain only (VHH) polypeptide containing an amino acid sequence having at least 85% sequence identity to a full-length amino acid sequence of any one of Tables 1, 6A to 6N, and 8, or a functional fragment thereof, and / or containing a set of Complementarity Determining Regions: CDR1, CDR2, and CDR3, selected from those listed in any one of Tables 2, 6A to 6N, and 8.

[0023] In another aspect, the disclosure features a VH-heavy chain only (VHH) polypeptide containing a full-length amino acid sequence listed in any one of Tables 1, 6A to 6N, and 8.

[0024] In another aspect, the disclosure features a chimeric antigen receptor polypeptide (CAR) containing the polypeptide of any aspect of the disclosure, or embodiments thereof.

[0025] In another aspect, the disclosure features a polynucleotide encoding the VHH polypeptide or the CAR of any aspect of the disclosure, or embodiments thereof.

[0026] In another aspect, the disclosure features a vector containing the polynucleotide of any aspect of the disclosure, or embodiments thereof.

[0027] In another aspect, the disclosure features a cell containing the polynucleotide or vector of any aspect of the disclosure, or embodiments thereof.

[0028] In another aspect, the disclosure features a pharmaceutical composition containing an effective amount of the polypeptide of any aspect of the disclosure, or embodiments thereof, or a CLDN18.1 or CLDN18.2 binding fragment thereof, or the CAR, the polynucleotide, the vector, or the cell of any aspect of the disclosure, or embodiments thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0029] In another aspect, the disclosure features a method for treating a subject having a neoplasia, the method involving administering to the subject the pharmaceutical composition of any aspect of the disclosure or embodiments thereof.

[0030] In another aspect, the disclosure features a kit containing the polypeptide, or a CLDN18.1 or CLDN18.2 binding fragment thereof, the CAR, the polynucleotide, the vector, the cell, or the pharmaceutical composition of any aspect of the disclosure, or embodiments thereof, and a container, for use in treating a subject having a neoplasia.

[0031] In another aspect, the disclosure features a method of detecting Claudin-18.1 (CLDN18.1) and / or Claudin-18.2 (CLDN18.2) polypeptide or a fragment thereof in a sample. The method involves contacting the sample with at least one detectably labeled VH-heavy chain only (VHH) polypeptide or an antigen binding fragment thereof that specifically binds to CLDN18.1 and / or CLDN18.2 or a fragment thereof. The VHH polypeptide or the antigen binding fragment thereof contains three Complementarity Determining Regions (CDRs), CDR1, CDR2, and CDR3 structurally positioned between four framework (FR) regions (FR1-FR4) as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; The three CDRs are selected from: CDR1 containing the amino acid sequence GRTF S SYA; CDR2 containing the amino acid sequence I S W SGGKT; and CDR3 containing an amino acid sequence selected from one or more of AAERDL L W LRYV VDY; VAERDL L W LRYWDY; AAERDLRW LRYWDY; AAERDL L W LRYWDD; AAERDL L L LRYWDY; AAERDL L W LRDWDY;

[0032] AAERDRL W LRYWDY; AAERN L L W LRYWDY; ATERDL L W LRYWDY; and AGERDL L W LRYWDY; CDR1 containing the amino acid sequence GRTF S SYI; CDR2 containing the amino acid sequence I S W SGDST; and CDR3 containing the amino acid sequence AAGRTTWANYAMDY; CDR1 containing the amino acid sequence GRTF S SYI; CDR2 containing the amino acid sequence I S W SGGST; and CDR3 containing the amino acid sequence AAGSTTWATYS MDY. Also, i) FR1 contains the following amino acid sequence: QQL VESGGGL X iQAGGS LRL SCAAS, where Xi is A or V; ii) FR2 contains the following amino acid sequence: MGW FRQAPGKEREX a VAX b, where Xais F or V; and Xb is A or G; iii) FR3 contains the following amino acid sequence:

[0033] YYX cDSX dKGRFTI SRDNXeKNTVYLQM N S LKXfEDTAVYYC, where Xcis A or D, Xdis V or L, Xeis A or G, Xf is T or P, and iv) FR4 contains the following amino acid sequence: WGQGTS VTV S S PTPAPTI. The method further involves, measuring the level of binding of the binding protein to the CLDN18.1 and / or CLDN18.2 polypeptide in the sample relative to a control to detect or identify the presence of CLDN18.1 and / or CLDN18.2 in the sample.

[0034] In another aspect, the disclosure features a method for identifying a VHH antibody that binds a target antigen. The method involves (a) preparing a library of expression vectors encoding chimeric antigen receptors (CARs), each containing a VHH domain generated in response to an antigen of interest. The method further involves (b) expressing each member of the library of expression vectors in a human primary T cell. The method also involves (c) contacting the human primary T cells with cells that do not express an antigen that is not the antigen of interest and sorting the human primary T cells into CD25+ and CD25- fractions and determining the sequences of VHH domains in each fraction to identify VHH domains enriched in the CD25- fraction. The method also involves (d) contacting the immune cell with cells expressing the antigen of interest, sorting the human primary T cells into CD25+ and CD25- fractions, and determining the sequences of VHH domains in each fraction to identify VHH domains enriched in the CD25+ fraction. The method also involves (e) identifying VHH domain sequences both enriched in the CD25+ fraction of (d) and in the CD25- fraction of (c), thereby identifying VHH domains that selectively bind the target antigen.

[0035] In another aspect, the disclosure features a method for identifying a VHH antibody that binds a target antigen. The method involves (a) preparing a library of expression vectors encoding chimeric antigen receptors (CARs), each containing a V HH domain generated in response to an antigen of interest. The method further involves (b) expressing each member of the library of expression vectors in a human primary T cell. The method also involves (c) contacting the immune cell with cells expressing the antigen of interest, sorting the human primary T cells into CD25+ and CD25- fractions, and determining the sequences of VHH domains in each fraction to identify VHH domains enriched in the CD25+ fraction, thereby identifying VHH domains that bind the target antigen.

[0036] In any aspect of the disclosure, or embodiments thereof, the VHH polypeptide or antigen binding portion thereof selectively binds to CLDN18.2.

[0037] In any aspect of the disclosure, or embodiments thereof, i) FR1 contains the following amino acid sequence: QQL VESGGGLXiQAGGS LRL SCAAS, where Xi is A or V; ii) FR2 cont ains the following amino acid sequence: MGW FRQAPGKEREX a VAX b, where Xais F or V; and Xb is A or G; iii) FR3 contains the following amino acid sequence: YYX cDSX dKGRFTI SRDNXeKNTVYLQM N S LKXfEDTAVYYC, where Xcis A or D, Xdis V or L, Xeis A or G, Xf is T or P, and iv) FR4 contains the following amino acid sequence:

[0038] WGQGTS VTV S S PTPAPTI.

[0039] In any aspect, or embodiments thereof, the disclosure features the CAR, where the CAR contains in order from N-terminus to C-terminus a CD28 signal peptide, the polypeptide, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 cytoplasmic domain, and a CD3( ^ domain.

[0040] In any aspect of the disclosure, or embodiments thereof, the vector is an expression vector.

[0041] In any aspect of the disclosure, or embodiments thereof, the cell is an immune cell. In any aspect of the disclosure, or embodiments thereof, the immune cell is a T cell. In any aspect of the disclosure or embodiments there, the cell is in vitro, e x vivo, or in vivo.

[0042] In any aspect of the disclosure, or embodiments thereof, the sample is selected from biopsy, blood, peripheral blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, tears, stool, or synovial fluid.

[0043] In any aspect, or embodiments thereof, the disclosure features a method where the CLDN18.1 or CLDN18.2 polypeptide or fragment thereof is on the surface of a cell.

[0044] In any aspect, or embodiments thereof, the disclosure features a method where the CLDN18.1 or CLDN18.2 polypeptide or fragment thereof is endogenously expressed by the cell.

[0045] In any aspect of the disclosure, or embodiments thereof, the neoplasia is a stomach cancer, a pancreatic cancer, an esophageal cancer, or an ovarian cancer.

[0046] In any aspect of the disclosure, or embodiments thereof, the VHH polypeptide, or functional fragment thereof, comprises a set of Complementarity Determining Regions: CDR1, CDR2, and CDR3, listed in a row of any one of Tables 2, 6A to 6N, and 8.

[0047] Definitions

[0048] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the various aspects and embodiments of the disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 198 8); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0049] By “agent” is meant any small molecule chemical compound, nucleic acid molecule, or polypeptide, or fragments thereof, or cell. In various embodiments, the agent is a VHH polypeptide of the disclosure or a polynucleotide encoding the VHH polypeptide (e.g., a VHH polypeptide capable of binding CLDN18.1 and / or CLDN18.2). In some embodiments, the agent is a chimeric antigen receptor expressing T cell (CAR-T cell).

[0050] By “alteration” is meant a change in the structure, sequence, expression levels, or activity of an analyte, clinical indicator, polynucleotide, or polypeptide as detected by standard art known methods, such as those described herein. The alteration can be an increase or a reduction.

[0051] By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0052] As used herein, the term “antibody” or “antigen-binding domain” refers to an immunoglobulin molecule, a nanobody, or a fragment thereof that specifically binds to, or is immunologically reactive with, a particular antigen. Non-limiting examples of antibodies or antigen-binding domains include V HH antibodies, polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including e.g., Fab', F (ab')2, Fab, Fv, rlgG, and scFv fragments, as well as engineered antibodies, which include CrossMabs (e.g., CrossMab Fabs, CrossMabCH1 'CLand CrossMabVH 'VLformats), or fragments thereof. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (such as, for example, Fab and F (ab')2 fragments) that are capable of specifically binding to a target protein. Fab and F (ab')2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non- specific tissue binding than an intact antibody (see Wahl et al., J. Nucl. Med. 24: 316, 1983; incorporated herein by reference).

[0053] Antibody structure is well known in the art. Briefly, the variable (V) regions or domains of antibody heavy (H) and light (L) chains contain Complementarity-Determining Regions (CDRs), which bind to specific antigens or immunogens (e.g., protein antigens or immunogens). CDRs are situated within framework (FR) sequences of the V regions of the heavy (VH) and light chains (VL) of an antibody. CDRs are the most variable parts of antibodies and are critical components in the diversity of antigen specificities of antibodies produced by B lymphocytes. In general, three CDRs (CDR1, CDR2 and CDR3) are arranged consecutively in a V domain of an antibody. Because a VHH, such as a camelid VHH, is essentially a single chain antibody polypeptide, it contains three CDRs that bind to an antigen or target protein such as human CLDN18.2 in the context of four framework (FR) regions, as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Because most of the sequence variability associated with immunoglobulins and antigen binding is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Typically, CDR1, CDR2 and CDR3 of VHHs contribute to and / or do not interfere with antigen binding. The CDRs and FR regions of a number of anti-CLDN18.2 VHHs described herein are shown, for example, in Tables 1, 2, 3 A, 3B, 7, and 8.

[0054] By “antigen” is meant an agent to which an antibody or other polypeptide capture molecule specifically binds. In an embodiment, the antigen is a tumor antigen. Exemplary antigens include small molecules, carbohydrates, proteins, and polynucleotides. In various embodiments, the antigen is CLDN18.1 or CLDN18.2. In some cases, an antibody or other polypeptide capture molecule specifically binds both CLDN18.1 and CLDN18.2.

[0055] A “camelid VHH framework region ( FR)” refers to the structural FR portions or components of a camelid VHH antibody or binding molecule, namely, FR1, FR2, FR3 and FR4, that positionally and structurally support the three CDR components, namely, CDR1, CDR2 and CDR3 of a VHH polypeptide, as described above. Similar to the FRs in conventional antibody polypeptides, the respective FR regions ( FR1, FR2, FR3 and FR4) of the anti-CLDN18.2 VHH polypeptides described herein are highly similar in sequence not only among different CLDN18.2 binding VHHs but also among camelid VHH polypeptides that bind to other antigens, e.g., unrelated VHH polypeptides. See, e.g., L. S. Mitchell and L. J. Colwell, 2018, Proteins, 86( 7): 697-706 and A. M. Vattekatte et al., March, 2020, PeerJ, 6( 8):e8 408. DOI: 10.7717 / peerj.8 408). Accordingly, the FR regions FR1, FR2, FR3 and FR4 of different V HHs do not vary significantly in sequence. By way of example, the below FR sequences of the VHH in the above-mentioned publication of Mitchell and Colwell are similar to the FR sequences of other VHHs, including the anti-CLDN18.2 VHH polypeptides described herein. Further exemplary FR sequences are listed in Tables 1 and 3.

[0056] FR1:

[0057]

[0058] FR1 (continued):

[0059]

[0060] FR3:

[0061]

[0062] FR3 (continued):

[0063]

[0064]

[0065] FR4:

[0066]

[0067] It will be appreciated that the amino acid position numbers of the VHH FRs shown above are approximate and may vary to some degree in length or amino acid sequence depending on VHH length and on the start and termination amino acid positions of the VHH CDRs. Thus, substantial similarities exist among the structural FRs of camelid VHHs, independent of antigen binding specificity.

[0068] A “chimeric antibody” refers to an antibody in which a region of an antibody of one species (e.g., rodent, mouse or rat) is replaced with that from another species (e.g., a human) to achieve a more human-like antibody. Chimeric antibodies may be recombinantly generated by combining the variable light and heavy chain regions obtained from antibody producing cells of one species with the constant light and heavy chain regions from another. In general, chimeric antibodies utilize rodent (or other species, such as rabbit or camelid) variable regions and human constant regions in order to produce an antibody with predominantly human constant domains. The production of chimeric antibodies is well known in the art, and may be achieved by standard means, for example, as described in U. S. Patent No. 5,624,659, incorporated fully herein by reference.

[0069] By “binding to” a molecule is meant having a physicochemical affinity for a molecule (e.g., a protein or protein antigen) or a region of the molecule, e.g., an epitope or antigenic determinant. Binding may be measured by any of the methods practiced in the art, e.g., using an antibody binding assay (e.g., ELISA) or an in vitro translation binding assay.

[0070] By “Chimeric Antigen Receptor” or alternatively a “CAR” is meant a polypeptide capable of providing an immune effector cell with specificity for a target cell. In embodiments, the target cell is a cancer cell. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule. In embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one embodiment the CAR comprises an optional leader sequence at the aminoterminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a VHH domain) during cellular processing and localization of the CAR to the cellular membrane. In various embodiments, the antigen binding domain comprises an anti-CLDN18 VHH antibody of the disclosure.

[0071] By “Claudin-18.1 (CLDN18.1) polypeptide” is meant a CLDN18.1 protein with at least about 85% amino acid sequence identity to NCBI Accession No. N P_057453.1, which is provided below, or a function fragment thereof. Representative functions of claudins (e.g., Claudin-18) are described in Krause, etal. “Structure and function of claudins, ” Biochimica et Biophysica Acta VIT '. 631-645 (2008), the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0072] > N P_057453.1 claudin-18 isoform 1 [Homo sapiens] M STTTCQWAF L L S I LGLAGCIAATGMDM W STQDLYDN P VTS V FQYEGL WRSCVRQS SGFTE CRPYFTI LGL PAM LQAVRAL M I VGI V LGAIGL L V S I FALKCIRIGS MEDSAKAN MTLTSGI M F I V SGLCAIAGV S V FAN M L VTN F W M STAN MYTGMGGM VQTVQTRYTFGAAL F VGW VAGGLTL IGGV M MCIACRGLAPEETNYKAV SYHASGH S VAYKPGGFKASTGFGS NTKNKKIYDGGARTE DEVQSYP SKHDYV

[0073] By “Claudin-18.1 (CLDN18.1) polynucleotide” is meant a nucleic acid molecule encoding an CLDN18.1 polypeptide, as well as the introns, exons, 3 ' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CLDN18.1 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CLDN18.1 expression. An exemplary CLDN18.1 nucleotide sequence from Homo Sapiens is provided below (NCBI Ref. Seq. Accession No.: NM_016369.4):

[0074] > NM_016369.4:62-847 Homo sapiens claudin 18 (CLDN18), transcript variant 1, mRNA ATGTCCACCACCACATGCCAAGTGGTGGCGTTCCTCCTGTCCATCCTGGGGCTGGCCGGCTG CATCGCGGCCACCGGGATGGACATGTGGAGCACCCAGGACCTGTACGACAACCCCGTCACCT CCGTGTTCCAGTACGAAGGGCTCTGGAGGAGCTGCGTGAGGCAGAGTTCAGGCTTCACCGAA TGCAGGCCCTATTTCACCATCCTGGGACTTCCAGCCATGCTGCAGGCAGTGCGAGCCCTGAT GATCGTAGGCATCGTCCTGGGTGCCATTGGCCTCCTGGTATCCATCTTTGCCCTGAAATGCA TCCGCATTGGCAGCATGGAGGACTCTGCCAAAGCCAACATGACACTGACCTCCGGGATCATG TTCATTGTCTCAGGTCTTTGTGCAATTGCTGGAGTGTCTGTGTTTGCCAACATGCTGGTGAC TAACTTCTGGATGTCCACAGCTAACATGTACACCGGCATGGGTGGGATGGTGCAGACTGTTC AGACCAGGTACACATTTGGTGCGGCTCTGTTCGTGGGCTGGGTCGCTGGAGGCCTCACACTA ATTGGGGGTGTGATGATGTGCATCGCCTGCCGGGGCCTGGCACCAGAAGAAACCAACTACAA AGCCGTTTCTTATCATGCCTCAGGCCACAGTGTTGCCTACAAGCCTGGAGGCTTCAAGGCCA GCACTGGCTTTGGGTCCAACACCAAAAACAAGAAGATATACGATGGAGGTGCCCGCACAGAG GACGAGGTACAATCTTATCCTTCCAAGCACGACTATGTGTAA

[0075] By “Claudin-18.2 (CLDN18.2) polypeptide” is meant a CLDN18.2 protein with at least about 85% amino acid sequence identity to NCBI Accession No. NP_001002026.1, which is provided below, or a function fragment thereof. Representative functions of claudins (e.g., Claudin- 18) are described in Krause, et al. “Structure and function of claudins,” Biochimica et Biophysica Acta VIT '. 631-645 (2008), the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0076] > NP_001002026.1 claudin- 18 isoform 2 [Homo sapiens] MAVTACQGLGF W S L IGIAGI IAATCMDQW STQDLYN N P VTAV F NYQGL WRSCVRES SGFTE CRGYFTL LGL PAM LQAVRAL M I VGI V LGAIGL L V S I FALKCIRIGS MEDSAKAN MTLTSGI M F I V SGLCAIAGV S V FAN M L VTN F W M STAN MYTGMGGM VQTVQTRYTFGAAL F VGW VAGGLTL IGGV M MCIACRGLAPEETNYKAV SYHASGH S VAYKPGGFKASTGFGS NTKNKKIYDGGARTE DEVQSYP SKHDYV

[0077] By “Claudin-18.2 (CLDN18.2) polynucleotide” is meant a nucleic acid molecule encoding an CLDN18.2 polypeptide, as well as the introns, exons, 3 ' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CLDN18.2 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CLDN18.2 expression. An exemplary CLDN18.2 nucleotide sequence from Homo Sapiens is provided below (NCBI Ref. Seq. Accession No.: NM_001002026.3):

[0078] > NM_001002026.3:54-839 Homo sapiens claudin 18 (CLDN18), transcript variant 2, mRNA ATGGCCGTGACTGCCTGTCAGGGCTTGGGGTTCGTGGTTTCACTGATTGGGATTGCGGGCAT CATTGCTGCCACCTGCATGGACCAGTGGAGCACCCAAGACTTGTACAACAACCCCGTAACAG CTGTTTTCAACTACCAGGGGCTGTGGCGCTCCTGTGTCCGAGAGAGCTCTGGCTTCACCGAG TGCCGGGGCTACTTCACCCTGCTGGGGCTGCCAGCCATGCTGCAGGCAGTGCGAGCCCTGAT GATCGTAGGCATCGTCCTGGGTGCCATTGGCCTCCTGGTATCCATCTTTGCCCTGAAATGCA TCCGCATTGGCAGCATGGAGGACTCTGCCAAAGCCAACATGACACTGACCTCCGGGATCATG TTCATTGTCTCAGGTCTTTGTGCAATTGCTGGAGTGTCTGTGTTTGCCAACATGCTGGTGAC TAACTTCTGGATGTCCACAGCTAACATGTACACCGGCATGGGTGGGATGGTGCAGACTGTTC AGACCAGGTACACATTTGGTGCGGCTCTGTTCGTGGGCTGGGTCGCTGGAGGCCTCACACTA ATTGGGGGTGTGATGATGTGCATCGCCTGCCGGGGCCTGGCACCAGAAGAAACCAACTACAA AGCCGTTTCTTATCATGCCTCAGGCCACAGTGTTGCCTACAAGCCTGGAGGCTTCAAGGCCA GCACTGGCTTTGGGTCCAACACCAAAAACAAGAAGATATACGATGGAGGTGCCCGCACAGAG GACGAGGTACAATCTTATCCTTCCAAGCACGACTATGTGTAA

[0079] In this disclosure, “comprises, ” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U. S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of ’ or “consists essentially” likewise has the meaning ascribed in U. S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments. The term “costimulatory molecule” refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD27, CD28, CDS, ICAM-1, LFA-1 (CDlla / CD18), ICOS (CD27 8), and 4-1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAF FR, HVEM (LIGHTR), SLAM F7, N Kp80 (KLRF1), N Kp44, N Kp30, N Kp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lid, ITGAE, CD 103, IT GAL, CD 11 a, LFA-1, ITGAM, CD11 b, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, N KG2D, N KG2C, TNFR2, TRANCE / RAN KL, DNAM1 (CD226), SLAM F4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAM F6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF 8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83.

[0080] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

[0081] By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an enzyme-linked immunosorbent assay (ELISA)), biotin, digoxigenin, or haptens.

[0082] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasias, such as tumors and cancers. In various instances, the cancer is a gastric cancer, a gastroesophageal adenocarcinoma, a pancreatic carcinoma, or a lung adenocarcinoma. In some embodiments, the disease is an epithelial-derived cancer. In some cases, the cancer is selected from one or more of stomach cancer, pancreatic cancer, esophageal cancer, and ovarian cancer.

[0083] By “effective amount” is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the various aspects and embodiments of the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In some embodiments, an effective amount induces killing of a cancer cell, reduces cancer cell survival, stabilizes tumor growth or cancer cell proliferation, or reduces tumor growth or cancer cell proliferation.

[0084] An “epitope tag” refers to a peptide or amino acid sequence (e.g., an epitope) that is fused, linked, or coupled to a protein, such as a recombinant protein produced by recombinant techniques, and that can be specifically bound by an antibody, e.g., an anti-tag monoclonal antibody or binding molecule that is directed to or generated against the tag peptide or amino acid sequence. Epitope tags are typically short peptide sequences (e.g., from about 5-30 amino acids, or sometimes up to 40 amino acids, that are selected because high-affinity antibodies can be reliably produced in many different species. Such antiepitope tag antibodies are optimally not cross-reactive with other human peptides or polypeptides and typically do not generate an antibody response, e.g., an anti-tag antibody response, when administered or delivered to a subject. An epitope tag sequence that is fused to a protein provides for the detecti o n a nd / or p u r ifica tion of the pr otein u sin g a n a n tibody, e. g., a m o n o c l o nal an t ibody, t h at, s pecif i c ally bi nds to t h e ep itop e t a g. I n a n embodi ment, t h e protein to which an e pito pe tag is fu sed, linked, or c o upled is a n antibody or VH H protein, e.g., a recombina n t ly produ ced an t ibody or VH H p rotein. In a n embodi men t, t he V HH i s a n an ti-CL D N18.2 an d / or anti-CLDN 18.1 VHH a n t ibody. In a n embo d i ment, the protein, may incl u d e on e o r m o re e pito pe t a gs. In a n e mbodim e nt, an epitope ta g is co u p l ed to the a m ino ( N H) ter m inu s of the pr o t e in, e.g., a VHH an t ibody a s des cribed her e in. I n a n embodiment, an epi top e ta g i s couple d to the c a rboxy (COOH) te r min u s of the p r o tein, e.g., a V HH an t ibody a s descr ibe d here i n. I n an e mbodi ment, a n e p itope tag i s c o u p led t o the N H a nd t h e CO OH termin i of the protei n, e.g., a VH H a ntibody a s describe d herein. An epit ope ta g sequ e n ce s uch as th ose des cribed her e i n may be bo und by an t i -ep i t o pe ta g ant ibodies, form i n g com plexe s which m ay fa cilit ate clea r a nce of the p rot ein c o n t aining the tags from the body o r sys tem. (See, a l s o, B. B riz zard and R. Ch ubet, 2001, Curr Pr o toc Neu r o sci., Ch a p ter 5, Un it 5. 8; DOI: 10.1002 / 0471 142301. n s 0508s00; R. Her n a n et a l., 2000, Biotech niques, 28(4):789-793; C. E. F ritze et al., 2000, Me ths Enzymol., 327:3- 16; doi: 10.1016 / s 0076-6879(00 )27263- 7; A. E i n hauer et al., 2001, J B i ochem B i ophys Methods, 49(l - 3 ):455- 65, d o i: 10.1 016 / s 0165~02 2x(01)00213-5)). Other mole c u l e s ma y s e rve a s protei n, a min o acid sequ e nce, o r polynu cle otide tags t hat are fused, linked, or coupled to a protein, such as a recombinant protein produced by recombinant techniques, e.g., an anti-CLDN18.2 and / or anti-CLDN18.1 VH H a n tibody des cribed here i n. I n a n embodi men t, t he tag can be specifically bound by an antibody, e.g., an anti-tag monoclonal antibody or binding molecule that is directed to or generated against the tag peptide or amino acid sequence. Exa mples of tags incl u d e, w ithout li m itati o n, FLAG ta g s (peptide sequen ce DYKDDDDK recognized by an anti-FLAG antibody), pol yHi s tidi ne (His ) ta g s (5-10 histidine residues ( H H H H H H) bound by a nickel or cobalt chelate), E-tag, a peptide comprising amino acid sequence GA P V PYPDP LEPR recognized by an antibody; an immunoglobulin Fc region or portion thereof, e.g., having effector or modulator function ( Fc tag). In particular, Fc tags comprise a domain (effector domain) of an immunoglobulin molecule, e.g., IgG, which can be genetically linked to a peptide or protein. Fc fusion proteins (also known as Fc chimeric fusion proteins, Fc-Igs, Ig-based chimeric fusion proteins, and Fc-tag proteins) are composed of an Ig Fc domain that is fused, linked, or coupled (e.g., by recombinant techniques) to a peptide or protein, such as an anti-CLDN18.2 or anti-CLDN18.1 VHH antibody described herein. The Fc domain portion of the fusion protein confers an advantageous characteristic to the protein, particularly in vivo, by greatly prolonging the half-life of the protein in plasma following administration to a subject. In an embodiment, an anti-CLDN18.2 or anti-CLDN-18.1 VHH antibody fused to an Fc region or Fc tag provides improved therapeutic efficacy as a biotherapeutic agent or drug. Fc fusion proteins also have uses in in vitro methods, including, e.g., immunohistochemistry (IHC), flow cytometry ( FC), protein binding assays and use as microarray baits. In these applications, the Fc domain serves as a support to which proteins can be attached while retaining their native biological activity. In addition, the Fc domain can improve the in vivo and in vitro solubility and stability of the protein or peptide molecule to which it is coupled, fused, linked, or attached.

[0085] By “ f ragment” is meant a portion of a polypeptide or nucleic acid molecule. In embodiments, portion contains, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0086] A “framework ( FR) region” or “FR region” includes amino acid residues that are adjacent to the CDRs in VH, and VL regions, and in VHHs. For example, FR region residues may be present in VHHs as described herein, camelid antibodies (VHHs), human antibodies, rodent-derived antibodies (e.g., murine and rat antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), VHHs, singlechain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others. Exemplary FR region amino acid sequences are provided in Tables 1 and 3.

[0087] The term “humanized” antibodies refers to forms of non-human (e.g., murine) antibodies, camelid-derived s i n g i e d o m ain a n t i b o d y ( sdAb) b i n d i n g m o l e c u les, w h i c h a re c o m p ris ed o f the he avy cha i n va r i abl e (V u ) r e gio n of he avy- chain - o nly a nti b o d i e s (Abs) or VHHs. Humanized antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F (ab')2 or other target-binding subdomains of antibodies) which contain minimal sequences derived from non-human immunoglobulin. In general, a humanized antibody or VHH may comprise substantially all of at least one variable domain (or two variable domains in the case of non-VHH antibodies), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All or substantially all of the FR regions of a humanized antibody may also be derived from a human immunoglobulin sequence. In the case of non-VHH antibodies, a VHH or a humanized antibody can also comprise at least a portion of an immunoglobulin constant region ( Fc), which may be that of a human immunoglobulin consensus sequence. Techniques and protocols for humanizing antibodies (as well as VHHs) are known and practiced in the art, as described, for examples, in Riechmann et al., Nature, 332:323-7, 198 8; Kasmiri et al., Methods, 36(l):25-34, 2005; U. S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and U. S. Patent No. 6,180,370 to Queen et al; EP239400; WO 1991 / 09967; U. S. Patent No.

[0088] 5,225,539; EP592106; and EP519596, the contents of which are incorporated herein by reference. Humanized antibodies or VHHs are molecularly engineered to contain even more human-like immunoglobulin domains, and incorporate only the CDRs of the VHH or animal-derived monoclonal antibody by carefully examining the sequence of the hyper-variable loops of the V regions of the monoclonal antibody or VHH, and fitting them to the structure of the human antibody chains. This process is routinely and commonly carried out by one having skill in the art. See, e.g., U. S. Patent No. 6,187,287, the contents of which are incorporated by reference herein.

[0089] By “increase” is meant to alter positively relative to a reference. An increase may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

[0090] An “intracellular signaling domain,” refers to portion of a molecule that transduces a signal from the surface of the cell to the interior of the cell. The intracellular signaling domain generates a signal that promotes an immune effector function of a CAR containing cell, e.g., a CART cell. Examples of immune effector function, e.g., in a CART cell, include cytolytic activity and helper activity, including the secretion of cytokines.

[0091] The terms “isolated, ” “purified, ” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0092] By “isolated polynucleotide” is meant a nucleic acid molecule that is free of the genes which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0093] By an “isolated polypeptide” is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, and or at least 99%, by weight, a polypeptide of the disclosure. An isolated polypeptide of the disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0094] By “marker” is meant any clinical indicator, analyte, protein or polynucleotide having an alteration in expression level or activity that is associated with a condition, disease, or disorder. A non-limiting example of a marker is a CLDN18.2 or a CLDN18.1 polypeptide. A “nanobody” as used herein refers to a si n g le-dom a in a ntibody or V HH. F o r e xample, a n a nobody r efers to a n a n t ibo dy frag m e nt o r p ortion wh i c h co n tain s a s ing l e m o nomeric va r i able dom ain (VH) na t u r ally occu r r ing in the Camelidae family o r synthetically de rived f r om the h e avy ch ain of a n a ntibody. S u c h s i n g le-dom a i n bi ndi n g mo lec u les c o mbin e hi gh anti g en affi nity in the absence of compl emen t -dependen t or cell- medi ated cytoto xicity d ue to the lack of a constant (Fc ) r e gion in these mole c u les.

[0095] By “neoplasia” is meant a disease or disorder characterized by excess proliferation or reduced apoptosis. In embodiments, a neoplasia is a cancer or tumor. Illustrative neoplasms include breast cancer, esophageal cancer, head-and-neck cancer, pancreatic cancer, skin cancer, colorectal cancer, hepatocellular cancer, bladder cancer, bile duct cancer, luminal and non-luminal bladder cancer, basal bladder cancer, muscle-invasive bladder cancer, and nonmuscle-invasive bladder cancer, pancreatic cancer, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin’s disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, liver cancer, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In embodiments, the neoplasia may be colon adenocarcinoma (COAD), stomach adenocarcinoma (STAD), stomach cancer, and uterine corpus endometrial carcinoma (UCEC). In embodiments, the neoplasia may be a liquid tumor such as, for example, leukemia or lymphoma. In embodiments, the cancer is a colon, kidney, lung, pancreatic, renal (e.g., renal cell carcinoma or clear renal cell carcinoma), or skin cancer (e.g., a melanoma). In embodiments, the neoplasia is a mesothelioma, pancreatic adenocarcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, cervical cancer, endometrial cancer, colorectal cancer, gastric cancer, esophageal cancer, or synovial sarcoma. In various instances, the neoplasia is a gastric cancer, a gastroesophageal adenocarcinoma, a pancreatic carcinoma, or a lung adenocarcinoma.

[0096] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0097] By “operably linked” is meant the connection between regulatory elements and one or more polynucleotides (genes) or a coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A polynucleotide (gene or genes) or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the polynucleotide (gene or genes) or coding region is controlled or influenced by the regulatory elements. The one or more polynucleotides may be separated by spacers or linkers.

[0098] “Percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0099] By “polypeptide” or “amino acid sequence” is meant any chain of amino acids, regardless of length or post-translational modification. In various embodiments, the post- translational modification is glycosylation or phosphorylation. In various embodiments, conservative amino acid substitutions may be made to a polypeptide to provide functionally equivalent variants, or homologs of the polypeptide. In some cases, the various aspects of the disclosure embrace sequence alterations that result in conservative amino acid substitutions. In some embodiments, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the conservative amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Non-limiting examples of conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In various embodiments, conservative amino acid substitutions can be made to the amino acid sequence of the proteins and polypeptides disclosed herein.

[0100] By “reduce” is meant to alter negatively relative to a reference. A reduction may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

[0101] By “reference” is meant a standard or control condition. In embodiments, a reference cell is a cell that does not express one of the VHH polypeptides presented herein. In some embodiments, a reference is a non- VHH anti-CLDN18.2 antibody that does not bind or detectably bind CLDN18.1. In some cases, a reference is a non- V HH anti-CLDN18.1 antibody. In some cases, a reference is a non- V HH anti-CLDN18.1 antibody that also binds CLDN18.2.

[0102] A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 10 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 35 amino acids, at least about 50 amino acids, or at least about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, or at least about 300 nucleotides, or any integer thereabout or therebetween. In embodiments, a reference sequence is a V HH antibody or a fragment thereof that does not bind a target antigen. In some cases, a reference sequence is a VHH antibody or a fragment thereof that binds a target antigen.

[0103] By “specifically binds” is meant recognizes and binds a polypeptide of the disclosure, but which does not substantially recognize and bind other molecules in a sample. In embodiments, a capture molecule is a VHH domain or a fragment thereof. A VHH domain or fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, a VHH domain or fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). A V HH domain or fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD of greater than 100 nM (e.g., greater than 500 nm, 1 pM, 100 pM, 500 pM, or 1 mM) for that particular antigen or epitope thereof. In some cases, an anti-CLDN18.2 VHH antibody is capable of specifically binding CLDN18.2 and does not bind or detectably bind CLDN18.1.

[0104] Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity.

[0105] Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (198 7) Methods Enzymol.

[0106] 152:399; Kimmel, A. R. (198 7) Methods Enzymol. 152:507).

[0107] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, about less than about 500 mM NaCl and 50 mM trisodium citrate, or about less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, of at least about 37° C, or of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0108] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, or of at least about 68° C. In one embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.

[0109] Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0110] By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In embodiments, such a sequence is at least 60%, at least 80% or 85%, or at least about 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0111] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e' 3and e'100indicating a closely related sequence.

[0112] By “subject” is meant an animal. The animal may be a mammal. The mammal may be a human or non-human mammal, such as a bovine, equine, canine, ovine, rodent, or feline.

[0113] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0114] “Transduction” refers to a process by which a polynucleotide is introduced or transferred into a cell. In an embodiment, the DNA or polynucleotide transduced into a cell is stably expressed in the cell. In some cases, the virus or virus vector is said to infect a cell.

[0115] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0116] As used herein, the term “vector” refers to a means of introducing a nucleic acid molecule into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, lipid nanoparticles, and episomes. Non-limiting examples of vectors include plasmids and cosmids. The term “vector” may refer to a nucleic acid (polynucleotide) molecule into which foreign nucleic acid can be inserted without disrupting the ability of the vector to be expressed in, replicate in, and / or integrate into a host cell. A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. A vector may contain a polynucleotide sequence that includes gene of interest (e.g., a heterologous gene, such as a therapeutic gene, or a reporter gene) as well as, for example, additional sequence elements capable of regulating transcription, translation, and / or the integration of these polynucleotide sequences into the genome of a cell. A vector may contain regulatory sequences, such as a promoter, e.g., a subgenomic promoter, region, and an enhancer region, which direct gene transcription. A vector may contain polynucleotide sequences (enhancer sequences) that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements may include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and / or a polyadenylation signal site to direct efficient transcription of a gene carried on the expression vector. Vectors, such as the viral particles described herein, may also be referred to as expression vectors.

[0117] As used herein, the term “vehicle” refers to a solvent, diluent, or carrier component of a pharmaceutical composition.

[0118] By “viral particle” is meant an agent capable of infecting a cell and that exists as an independent particle containing a core viral genome or polynucleotide, a capsid, which surrounds the genetic material and protects it, and an envelope of lipids surrounding the capsid. A viral particle may refer to the form of a virus before it infects a cell and becomes intracellular, or to the form of the virus that infects a cell.

[0119] By “VHH domain” is meant an antigen binding domain of a heavy chain only antibody or an antigen binding fragment thereof.

[0120] A “VHH binding molecule” or “VHH antibody,” or simply “VHH,” as referred to herein is, in general, a single domain immunoglobulin molecule (antibody). A V HH (or VHH antibody) corresponds to the heavy chain of a VHH antibody having a single variable domain (or single variable region), e.g., a camelid-derived single variable H (VH) domain antibody. A V HH typically has a molecular weight (MW) of about 12-15 kDa. VHH antibodies lack light chains. These heavy-chain antibody molecules contain a single variable domain (VHH) and, typically, two constant domains (CH2 and CH3). See, e.g., Methods in Molecular Biology, “Single Domain Antibodies - Methods and Protocols,” Eds. D. Saerens and S. Muyldermans, Humana Press (Springer), 2012. A cloned (recombinantly produced) and isolated V HH domain is a stable polypeptide harboring the antigen-binding capacity of the original heavy-chain antibody. See, e.g., U. S. Patent No. 5, 840,526 and U. S. Patent No.

[0121] 6,015,695, each of which is incorporated by reference herein in its entirety. In various embodiments, the VHH antibody is an anti-CLDN18.1 or anti-CLDN18.2 VHH antibody of the disclosure.

[0122] VHHs are efficiently expressed in E. coli, coupled to detection markers, such as a fluorescent marker, or conjugated with enzymes. The small size of VHHs permits their binding to epitopes (antigenic determinants in antigen proteins), e.g., “hidden epitopes” that are not accessible to whole antibodies of much larger size. As a therapeutic, a VHH is capable of efficient penetration and rapid clearance. Its single domain nature allows a VHH to be expressed in a cell without a requirement for supramolecular assembly, as is needed for whole antibodies which are typically tetrameric (two heavy chains and two light chains, having a MW of about 150 kDa). VHHs are also exhibit stability over time and have a longer half-life versus non- VHH antibody molecules, which comprise disulfide bonds that are susceptible to chemical reduction or enzymatic cleavage. Similar to immunoglobulins, VHHs may be modified post-translationally, e.g., to add chemical linkers, detectable moi eties, such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties, etc., or specific binding moieties, such as streptavidin, avidin, or biotin, etc., for use in the compositions and methods described herein.

[0123] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0124] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0125] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0126] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0127] BRIEF DESCRIPTION O F THE DRAWINGS FIGs. 1A to ID provide flow cytometry histograms and a schematic diagram. FIG.

[0128] 1A provides a flow cytometry histogram demonstrating that >85% of DC2.4 cells administered to nanomice surface expressed CLDN18.2. FIG. IB provides a schematic diagram of a timeline for vaccinating mice with CLDN18.2+ DC2.4 cells. CLDN18.2 was introduced into DC2.4 cells through lentiviral transduction using a lentiviral vector containing a polynucleotide encoding a CLDN18.2 antigen. CLDN18.2 antigens expressed in the DC2.4 cells were presented by MHC class I and / or class II molecules on the surface of the cells. The DC2.4 cells were stimulated with CpG oligodeoxynucleotides for about 4 hours prior to being used to vaccinate the nanomice with about 5 million cells by intraperitoneal injection (IP) and subcutaneous injection. After about two weeks, the animals were administered a boost vaccination. After about two additional weeks, cells (e.g., antibodyproducing B cells) were harvested from the nanomice. FIG. 1C provides a flow cytometry histogram demonstrating that immunolabeled LentiX cells with serum isolated from nanomice vaccinated with DC2.4_CLDN18.2 cells positively expressed CLDN18.2. FIG. ID provides a stacked flow cytometry histogram demonstrating that nanomice (M8, M6, M7, etc.) vaccinated with only CLDN18.2+ cells produced anti-CLDN18.2 VHH antibodies, while nanomice vaccinated with only MSLN+ cells (a negative control) did not. In FIG. 1C, the term “MSLN ” refers to mesothelin. The VHH antibodies of the present disclosure originated from the vaccinated mice referred to as M9 and Ml 1 in FIG. 1C. Some of the vaccinated mice produced no or only low or undetectable levels of anti-CLDN18.2 VHH antibodies.

[0129] FIG. 2 provides a plot showing clustering of VHH antibody sequences of the disclosure. The Starred data points indicate sequences corresponding to the indicated VHH antibody.

[0130] FIGs. 3 A to 3E provide bar graphs and a schematic diagram. FIG. 3 A provides a bar graph demonstrating that Jurkat cells transduced with libraries of chimeric antigen receptors (CARs) containing as their antigen binding domains V HH domains from nanomice vaccinated according to the method described in FIG. IB (“vaxlibs” or “vaccination libraries”) were activated when cultured in the presence of target K562 cells surface expressing CLDN18.1 or CLDN18.2 but not when cultured in the presence of MUC16.

[0131] MUC16, which refers to mucin- 16, served as a mis-matched control antigen. In FIGs. 3 A, 3C, and 3 D, the term “unmodified” refers to T cells that did not express a chimeric antigen receptor. Jurkat cells expressing vaccination libraries 9 and 11 demonstrated increased activation and were further characterized (see FIGS. 3C and 3 D). FIG. 3B provides a schematic diagram showing a protocol for preparation and characterization of the vaccination libraries. FIGS. 3C and 3 D provide bar graphs demonstrating the activation potential of specific chimeric antigen receptors (e.g., 9.4, 9.11, 9.14, 9.20, 9.23, 9.24, 9.28, 11.1, 11.2, 11.4, 11.5, 11.6, 11.9, 11.10, 11.11, 11.13, 11.14, and 11.15) from vaccination libraries 9 and 11, respectively. FIG. 3E provides a bar graphs demonstrating that Jurkat cells surfaceexpressing chimeric antigen receptors (CARs) containing anti-CLDN18.2 VHH domains provided herein as antigen binding domains were activated when cultured in the presence of target K562 cells surface-expressing CLDN18.1, CLDN18.2, or MUC16. FIG. 3E provides a plot of the fold change of target K562 cells transgenically expressing CLDN18.1, CLDN18.2, or MUC16 that were co-cultured with Jurkat cells expressing chimeric antigen receptors (CARs) containing as their antigen binding domains a VHH antibody selected from 9.4, 11.05, 11.11, and 11.13. The Jurkat cells were co-cultured with the target K562 cells at an effector-to-target (E: T) ratio of about 1:1.

[0132] FIG. 4 provides a flow cytometry histogram demonstrating that the anti-CLDN18.2 VHH antibody 9.4 selectively bound CLDN18.2.

[0133] FIGs. 5A and 5B provide maps for a plasmid used in the Examples to express chimeric antigen receptors containing a VHH domain of the disclosure. These plasmids were used to prepare vaccination libraries. FIG. 5A provides a map for the full plasmid and FIG.

[0134] 5B provides a map for the portion of the plasmid encoding a chimeric antigen receptor linked to enhanced green fluorescent protein (eGF P) by way of a P2A self-cleaving peptide.

[0135] FIGs. 6A and 6B provide maps for a plasmid used in the Examples to detect activation of chimeric antigen receptor (CAR) T cells. FIG. 6A provides a map for the full plasmid, and FIG. 6B provides a map of the portion of the plasmid corresponding to a reporter cassette.

[0136] FIG. 7 provides a schematic diagram providing an overview of an in vitro method for screening V HH domain libraries to identify VHH domains capable of binding to an antigen of interest (e.g., CLDN18.2 or CLDN18.1).

[0137] FIG. 8 provides a set of flow cytometry contour plots showing that many human primary T cells transduced with libraries of chimeric antigen receptors (CARs) containing as their antigen binding domains V HH domains from vaxlib 9 showed activation, as measured by CD25 immunostaining, when contacted with K562 cells (target cells) expressing CLDN18.2. Levels of CD25 immunostaining increased with each stimulation with the K562 cells. The flow cytometry countour plots labeled “Day 3” represents measurements taken after a 1 ststimulation, and the flow cytometry heat maps labeled “Day 5” represents measurements taken after a 2ndstimulation. In FIG. 8, “UTD” indicates target cells that did not express CLDN18.1 or CLDN18.2, “CLDN18.1” indicates target cells that expressed CLDN18.1, and “CLDN18.2” indicates target cells that expressed CLDN18.2. FIG. 9 provides plots demonstrating recovery of VHH domains from each screening condition. In FIG. 9, 910, 94, 911, etc. represent the VHH domains CLDN 9.10, CLDN 9.4, CLDN 9.11, etc. The cells screened were human primary T cells transduced with libraries of chimeric antigen receptors (CARs) containing as their antigen binding domains VHH domains from vaxlib 9 (“M9 library cells”). In FIG. 9, “m9inpuf ’ references the library composition (i.e., vaxlib 9) input to the screens, “m9TcellCD25neg” indicates M9 library cells showing lack of activation when not co-cultured with other cells, “m9utdCD25neg” indicates M9 library cells input cells showing lack of activation when co-cultured with K562 cells, “m9CLDN18vlCD25neg” indicates M9 library cells showing lack of activation when co-cultured with K562 cells expressing CLDN18.1, “m9CLDN18v2CD25neg” indicates M9 library cells showing lack of activation when co-cultured with K562 cells expressing CLDN18.2, and “m9CLDNv2CD25pos” indicates M9 library cells showing activation when co-cultured with K562 cells expressing CLDN18.2. In FIG. 9, “CPM” represents counts per million. In FIG. 9 the sequence abundance from each in vitro screen condition (Y-axis) was correlated with the original vaccination repertoire from vaxlib 9 (X-axis) to ensure adequate sequence capture.

[0138] FIG. 10 provides a plot showing V HH domains enriched from vaxlib 9 in screens carried out as described in FIG. 7. In FIG. 10, 94, 914, 97, etc. represent the V HH domains CLDN 9.4, CLDN_9.14, CLDN_9.7, etc. In FIG. 10 “loglOFC CD25+ vs CD25-” represents the log base 10 enrichment of a particular VHH domain in a screen selecting for activation in the presence of target cells expressing CLDN18.2 relative to the abundance of the V HH domain in a screen selecting for lack of activation in the presence of target cells expressing CLDN18.2. V HH domains corresponding to a “hit score”, which was calculated as described in the Examples provided below, of greater than 0.5 were considered hits. The x-axis of FIG. 10 represents the frequency of each V HH domain in vaxlib 9 (Mouse 9) prior to screening. In FIG. 10, the size of each data point is proportional to statistical significance.

[0139] FIG. 11 provides a t-distributed stochastic neighbor embedding plot (t-SN E) of the V HH domain amino acid sequences corresponding to FIG. 10. The “hit score” referenced in FIG. 11 is the same “hit score” as FIG. 10. In FIG. 11, the sequences analyzed were CDR3 sequences of the V HH domains.

[0140] FIG. 12 provides CDR3 sequences for V HH domains corresponding to the indicated group from FIG. 11. The following CDR3 amino acid sequences are listed in FIG. 12:

[0141] A AERDL L W LRYWDY; V AERDL L W LRYWDY; T YAERDLRW LRYWDY; AAERDL L W LRYWDD; AAERDL L L LRYWDY; AAERDL L W LRDV VDY;

[0142] AAERDRL W LRYWDY; AAERN L L W LRYV VDY; ATERDL L W LRYWDY; and AGERDL L W LRYWDY.

[0143] FIGs. 13A and 13B provide an alternative presentation of the plot of FIG. 11 where VHH domains corresponding to particular data points are identified (FIG. 13B) (e.g., 924, 97, 911, etc. which correspond to VHH domains CLDN_9.24, CLDN_9.7, CLDN_9.11, etc., respectively) or each data point is identified as corresponding to a “hit” (FIG. 13A), where a “hit” is as described above for FIG. 10.

[0144] FIG. 14 provides a plot demonstrating the in vivo killing of target tumor cells (the GSU stomach adenocarcinoma cell line) by human primary T cells expressing a chimeric antigen receptor (CAR) containing as its antigen binding domain the VHH domain CLDN 9.4.

[0145] DETAILED DESCRIPTION

[0146] Described herein are single domain antibody (sdAb) binding molecules, which are comprised of the heavy chain variable (VH) region of heavy-chain-only antibodies (Abs), that bind to a CLDN18.2 and / or CLDN18.1 polypeptide. In some cases, the sdAB binding molecules selectively bind CLDN18.2. In some embodiments, the present disclosure provided chimeric antigen receptors (CAR) comprising a VHH polypeptide of the disclosure as an antigen binding domain, and cells (e.g., CAR-T cells) expressing the same.

[0147] The various aspects and embodiments of the disclosure are based, at least in part, upon the discovery detailed in the Examples provided herein of VHH antibodies capable of binding to CLDN18.1 or CLDN18.2. Single-domain antibodies (camelid single-domain antibodies or nanobodies) are called VHHs as they derive from the VH region of a class of heavy-chain-only antibodies. The anti-CLDN18.2 or anti-CLDN18.1 VHHs were produced from immunized nanomice and were selected for their ability to bind to CLDN18.2 or CLDN 18.1. The anti-CLDN 18.2 or CLDN 18.1 VHHs as described herein are polypeptides comprising hypervariable variable regions (CDRs) within framework ( FR) regions. In general, the FRs of VHHs are typically highly similar in amino acid sequence or differ by conservative amino acid substitutions at certain positions of the FR sequences.

[0148] The anti-CLDN18.2 or anti-CLDN18.1 VHH antibodies as described herein provide advantageous properties, particularly for therapeutic use. By way of nonlimiting example, these sdAb molecules are small proteins (e.g., about 27 Kda), thus facilitating the cloning of their encoding polynucleotides. In various embodiments, the anti-CLDN18.2 or anti-CLDN18.1 VHHs can be functionally expressed at high levels, are stable to extreme pH and high temperatures over time, and function well in multimeric forms, e.g., dimers and other multimers, to provide improved binding and neutralization properties and therapeutic efficacy.

[0149] Chimeric antigen receptor (CAR) T cells expressing chimeric antigen receptors containing VHHs of the disclosure may be employed as therapeutic agents for the treatment and prevention of CLDN18.2-mediated or CLDN 18.1 -mediated and associated disorders, conditions, or diseases as described herein. In embodiments, a VHH of the disclosure is conjugated to a cytotoxic agent capable of killing a CLDN18.2 or CLDN18.1 expressing neoplastic cell. It will be understood that the terms “anti-X VHH antibody, ” “anti-X VHH polypeptide, ” “anti-X VHH antibody polypeptide,” “anti-X VHH,” and “XVHH” are used interchangeably herein, where “X” may be CLDN18.2 or CLDN18.1.

[0150] Cl audin

[0151] Claudins are a family of proteins in mammals consisting of four-pass transmembrane proteins expressed at the tight junction of epithelial and endothelial cells. There are at least 27 members of the Claudin family ranging between 20-27 kDA. Claudins regulate signal transduction and transport of selective molecules past epithelial cells. Claudin- 18, also known as CLDN18, is a protein that in humans is encoded by the CLDN18 gene. The Claudin-18.2 (CLDN18.2) and Claudin-18.1 (CLDN18.1) isoform variants of Claudin-18 arise from the alternative splicing of CLDN18 exonlb / la, respectively. CLDN18.2 is markedly different from CLDN18.1 in the N-terminal 69 amino acids, and by approximately 8 amino acids in the first extracellular loop.

[0152] Transcription profiling of restricted sets of tissues have previously revealed CLDN18.1 displays specificity in healthy lung tissue, whereas CLDN18.2 is predominantly expressed in healthy stomach tissue and is aberrantly upregulated in malignant cancer cells of, e.g., the stomach, pancreas, esophagus, and ovaries. In healthy humans, CLDN18.2 expression is tightly restricted to stomach tissue. Activation of CLDN18.2 depends on transcription factor cyclic AMP-responsive element binding protein (CREB) binding to its unmethylated consensus site. CLDN18.2 is upregulated in several human malignancies, including esophageal, pancreatic adenocarcinomas, non-small cell lung, and gastric, and is retained on malignant transformation. Since CLDN18.2 is overexpressed in several cancers, the protein may be exploited as tumor marker that may be detected using polypeptides of the present disclosure. Further, the present disclosure provides methods for treating a subject having a malignancy associated with over-expression of CLDN18.2. The methods may involve administering a polypeptide of the disclosure or antigen-binding fragment thereof, or a polynucleotide encoding the same, or a cell expressing a chimeric antigen receptor (CAR) containing the polypeptide or antigenbinding fragment thereof to the subject.

[0153] VHH Antibodies

[0154] In various aspects, the disclosure provides VHH antibodies, also known as “nanobodies, ” capable of binding or selectively binding a CLDN18.2 or CLDN18.1 antigen, as well as polypeptides containing VHH domains or polynucleotides encoding the same. In embodiments, the CLDN18.2 or CLDN18.1 polypeptide bound by the VHH antibodies is associated with a disease or disorder, such as a neoplasia. In embodiments, the VHH binds an antigen associated with a target cell. In embodiments, the target cell is a neoplastic cell.

[0155] VHH domains are derived from nanobodies. Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, a cloned and isolated VHH domain is a stable polypeptide harboring the full antigen-binding capacity of the original heavy-chain antibody. Nanobodies have a high homology with the VH domains of human antibodies and can be further humanized without any loss of activity. Importantly, Nanobodies have a low immunogenic potential, which has been confirmed in primate studies with Nanobody lead compounds.

[0156] Nanobodies combine the advantages of conventional antibodies with important features of small molecule drugs. Like conventional antibodies, nanobodies show high target specificity, high affinity for their target, and low inherent toxicity. However, like small molecule drugs they can inhibit enzymes and readily access receptor clefts. Furthermore, Nanobodies are stable, can be administered by means other than injection (see, e.g., W02004041867A2, which is herein incorporated by reference in its entirety) and are easy to manufacture. Other advantages of Nanobodies include recognizing uncommon or hidden epitopes as a result of their small size, binding into cavities or active sites of protein targets with high affinity and selectivity due to their unique 3 -dimensional, drug format flexibility, tailoring of half-life and ease and speed of drug discovery. Nanobodies are encoded by single genes and are efficiently produced in almost all prokaryotic and eukaryotic hosts, e.g., E. coli (see, e.g., U. S. Pat. No. 6,765,087, which is herein incorporated by reference in its entirety), molds (for example Aspergillus or Trichoderma) and yeast (for example Saccharomyces. Kliiyveromyces. Hansenu la. or Pichid ) (see, e.g., U. S. Pat. No. 6,838,254, which is herein incorporated by reference in its entirety).

[0157] VHHs, such as the anti-CLDN18.2 and / or anti-CLDN18.1 VHHs described herein, have a number of advantages over conventional antibodies and recombinant antibody domains, including (i) they are small monomeric proteins (27 kDa) that express and fold efficiently in recombinant hosts; (ii) they are more stable to extremes of pH and temperature compared with conventional antibodies; (iii) they typically bind conformational epitopes; and (iv) they are amenable to designed multimerization which often leads to higher potencies; and (v) they offer more therapeutic versatility, such as multispecificity, thus supporting their beneficial utility in treating diseases caused by or associated with CLDN18.2 or CLDN18.1.

[0158] The amino acid sequences of representative anti-CLDN18.2 VHH and anti-CLDN18.1 antibodies described herein are provided in Table 1 below. Representative embodiments of the binding regions of the anti-CLDN18.2 VHHs and anti-CLDN18.1 VHHs include CDRs (CDR1, CDR2 and CDR3) as set forth in Table 2 below. The CDRs are positioned within framework ( FR) regions of the VHH polypeptide (see Table 3), which do not vary substantially in sequence between discrete VHHs and which provide a “structural scaffold” for the CDRs, which bind to an antigen. By way of non-limiting example, the binding of CDRs within FRs to a target protein (antigen), e.g., CLDN18.2, may be via conformational binding or interaction, electrostatic binding interaction, hydrogen bonding, Van der Waals forces, or hydrophobic bonding, or combinations thereof, as would be appreciated by those having skill in the art. In various embodiments, the CDR3 of CLDN 9.4 contains an amino acid sequence selected from one or more of the following:

[0159] AAERDL L W LRYV VDY; VAERDL L W LRYWDY; T YAERDLRW LRYWDY;

[0160] AAERDL L W LRYV VDD; T YAERDL L L LRYWDY; T YAERDL L W LRDWDY;

[0161] AAERDRL W LRYWDY; T YAERN L L W LRYWDY; ATERDL L W LRYWDY; and AGERDL L W LRYWDY.

[0162] Table 1. Anti-CLDN18 VHH antibody amino acid sequences. Representative CDRs are underlined, and rep resentative FR regio ns are shown as plain text.

[0163]

[0164]

[0165] Table 2. Representative embodiments of complementarity determining regions of anti-CLDN18 VHH antibodies of the disclosure

[0166]

[0167] Table 3. F ramewo rk regio ns of anti-CLD N18 VH H antibodies of the disclosure

[0168]

[0169] Table 3 (co n tinued)

[0170]

[0171] In various embodiments, the FR1 of a VHH antibody of the disclosure contains the following amino acid sequence:

[0172] QQLVESGGGLXiQAGGSLRLSCAAS, wherein

[0173] Xi is A or V.

[0174] In various embodiments, the FR2 of a VHH antibody of the disclosure contains the following amino acid sequence:

[0175] MGWFRQAPGKEREXaVAXb, wherein

[0176] Xais F or V; and

[0177] Xb is A or G.

[0178] In various embodiments, the FR3 of a VHH antibody of the disclosure contains the following amino acid sequence:

[0179] YYXcDSXdKGRFTISRDNXeKNTVYLQMNSLKXfEDTAVYYC, wherein Xcis A or D;

[0180] Xais V or L;

[0181] Xeis A or G; and

[0182] Xf is T or P.

[0183] In various embodiments, the FR4 of a V HH antibody of the disclosure contains the following amino acid sequence: WGQGTS VTV S S PTP A PTI.

[0184] The CDRs of the anti-CLDN18.2 V HH or anti-CLDN18.1 polypeptides described herein may vary in amino acid sequence length. By way of nonlimiting example, CDR1 of the anti-CLDN18.2 V HH or anti-CLDN18.1 polypeptides as described herein may comprise from about 5 to about 12 (e.g., 5 or 8) amino acid residues; CDR2 may comprise from about 7 to about 20 (e.g., 16, 17, or 8) amino acid residues; and CDR3 may comprise from about 5 to about 25 (e.g., 7-13, 15-24, or 16) amino acid residues. It will be appreciated by one skilled in the art that number of amino acids that constitute a CDR is not necessarily precise. In some cases, an amino acid residue, or 2 or 3 amino acid residues, at one end or both ends of a given CDR may be considered as part of the CDR or as part of the neighboring FR region. The CDR regions of representative anti-CLDN18.2 VHH and anti-CLDN18.1 antibody polypeptides described herein are presented in Table 2. The anti-CLDN18.2 VHH and anti-CLDN18.1 antibodies described herein demonstrate the CDR diversity that is selected during affinity maturation of CLDN18.2 or CLDN18.1 binding polypeptides in the same animal. Despite such CDR diversity, the CLDN18.2 or CLDN18.1 binding VHHs generated as described herein show detectable binding to CLDN18.2 and / or CLDN18.1. The anti-CLDN18.2 or anti-CLDN18.1 VHH polypeptides demonstrate significant binding to the CLDN18.2 or CLDN18.1 antigen, despite some variation among the CDR sequences in the context of their framework regions.

[0185] In view of the representative anti-CLDN18.2 and anti-CLDN18.1 VHH amino acid sequences listed in Table 1, it will be appreciated by one skilled in the art that individual VHH polypeptides, (e.g., of from about 105 to about 140 amino acids in length and comprising 3 CDRs and 4 FR regions), which comprise at least about or equal to 85%, or 8 8%, or greater identity in amino acid sequence bind to the CLDN18.2 or CLDN18.1 antigen. In an embodiment, at least about or equal to 85%, 86%, 8 7%, 8 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity is tolerated among the anti-CLDN18.2 or anti-CLDN18.1 VHHs without adversely affecting or eliminating binding of the VHH polypeptides to the CLDN18.2 or CLDN18.1 antigen. In an embodiment, such amino acid sequence variation among the anti-CLDN18.2 or anti-CLDN18.1 VHH polypeptides is tolerated in the CDRs of the VHH polypeptides without adversely affecting binding of the VHHs to CLDN 18.2 or CLDN 18.1. In a particular embodiment, the amino acid sequence variations between or among anti-CLDN18.2 or anti-CLDN18.1 VHHs encompass one or more conservative amino acid substitutions or changes in a VHH amino acid sequence. In an embodiment, the one or more conservative amino acid substitutions or changes in a VHH amino acid sequence occur in one or more CDR sequences of the VHH, in one or more FR sequences of the VHH, or in CDR and FR sequences of the VHH.

[0186] The three CDRs of the anti-CLDN18 VHH polypeptides are arranged or positioned in the context of four FR regions as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to the framework regions 1-4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1-3, respectively. Similar to the FRs in conventional antibody polypeptides, the respective FRs (FR1, FR2, FR3 and FR4) of the anti-CLDN18.2 or anti-CLDN18.1 VHH polypeptides described herein are highly similar in sequence among different VHHs that were generated. Accordingly, provided are anti-CLDN18.2 or anti-CLDN18.1 VHH polypeptides comprising CDR1-3, in the structural context of FR1-4, that bind to a CLDN18.2 or CLDN18.1 protein, or to suitable fragments of the CLDN18.2 or CLDN18.1 protein, as well as polypeptides that comprise or consist essentially of one or more of the anti-CLDN18.2 or anti-CLDN18.1 VHHs and / or CLDN18.2 or CLDN18.1 binding fragments thereof (e.g., a chimeric antigen receptor (CAR) polypeptide).

[0187] In addition, the FRs of the CLDN18.2 -binding VHHs described herein are highly or essentially similar in sequence to the FRs of VHHs produced in camelid animals, such as alpacas, camels, llamas, and the like. As they provide structural and conformational support for the CDRs of V HH polypeptides, the FRs and the FR1, FR2, FR3 and FR4 regions among camelid VHH polypeptides generally share significant sequence identity. See, e.g., A. M. Vattekatte et al., March, 2020, PeerJ., 6(8):e8408. DOI: 10.7 717 / peerj.8408 and L. S. Mitchell and L. J. Colwell, 2018, Proteins, 86(7): 697-706).

[0188] Table 3 presents the amino acid sequences of the four framework regions, i.e., FR1, FR2, FR3 and FR4, respectively, of representative VHH polypeptides described herein. The alignment of the sequences supports substantial similarity among the structural FRs of the anti-CLDN18.2 camelid VHH antibodies described herein.

[0189] In embodiments, in cases in which a FR (or CDR) amino acid residue in a VHH polypeptide may be one of several alternative amino acid residues, the alternative amino acid residues will frequently share similar characteristics or properties, e.g., hydrophobicity, polarity, and / or charge. A conservative replacement (also called a conservative substitution) is an amino acid replacement or substitution in a polypeptide or region thereof that changes a given amino acid residue to a different amino acid residue with similar biochemical properties, such as charge, hydrophobicity, and / or size. By way of non-limiting example, the below Table 4 presents amino acids and their 1 -letter codes categorized into six main classes based on their structure and the general chemical characteristics of their side chains (R groups).

[0190] Table 4. Classes of amino acids based on structural and chemical characteristics of their side chains. In embodiments, an amino acid within an FR or CDR of a VHH antibody of the disclosure is substituted with another amino acid from the same class indicated in Table 4.

[0191]

[0192] In an embodiment, amino acid sequence substitutions or changes in an anti-CLDN18.2 or anti-CLDN18.1 VHH polypeptide relative to another anti-CLDN18.2 or anti-CLDN18.1 VHH polypeptide comprise conservative amino acid substitutions or changes such that a given amino acid residue is substituted with or replaced by a different amino acid residue with similar biochemical properties, such as charge, hydrophobicity, and / or size. In an embodiment, sequence variation between or among anti-CLDN18.2 or anti-CLDN18.1 VHH polypeptides results from one or more conservative amino acid changes and account for the percent sequence variation, e.g., 85%, 86%, 8 7%, 8 8%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence variation.

[0193] In some embodiments, the VHHs as described herein are humanized using methods and techniques practiced by those having skill in the art. (See, e.g., U. S. Patent Nos.

[0194] 8,975,382 and 10,550,174, the contents of which are incorporated by reference herein).

[0195] The anti-CLDN18.2 or anti-CLDN18.1 VHH antibodies described herein have widespread application (e.g., as antigen binding domains of chimeric antigen receptor polypeptides). In embodiments, the disclosure provides polynucleotides that encode operably linked modular components that constitute the described anti-CLDN18.2 or anti-CLDN18.1 VHHs. In embodiments, the anti-CLDN18.2 and anti-CLDN18.1 VHHs are recombinantly produced. In embodiments, the anti-CLDN18.2 or anti-CLDN18.1 VHHs encompass the proteins (polypeptides) encoded by the polynucleotides. In embodiments, the polynucleotide is DNA, cDNA, RNA, mRNA, or the like. In an embodiment, the anti-CLDN18.2 or anti-CLDN18.1 V HHs may be humanized or codon-optimized using methods practiced by those having skill in the art.

[0196] Suitable methods of producing or isolating antibody fragments having the requisite binding specificity and affinity for binding to an epitope tag include for example, methods which select recombinant antibody from a library or by PCR (e.g., U. S. Patent No. 5,455,030 and U. S. Patent No. 7,745,587 each of which is incorporated by reference herein in its entirety).

[0197] Functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered, or single chain antibodies, can also be produced. Functional fragments or portions of the foregoing antibodies include those which are reactive with the CLDN18.2 or CLDN18.1 protein. For example, antibody fragments capable of binding to CLDN18.2 or CLDN18.2, or a portion thereof, include, but not limited to, scFvs, Fabs, VHHs, Fv, Fab, ’ab' and F ’ab')2. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage are used generate Fab or F ’ab')2 antibody fragments, respectively. Antibody fragments are produced in a variety of truncated forms using antibody-encoding genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F ’ab')2 heavy chain peptide portion can be designed to include DNA sequences encoding the CHi peptide domain and hinge region of an immunoglobulin heavy chain.

[0198] Epitope ta gs

[0199] In certain embodiments, an anti-CLDN18.2 or anti-CLDN18.1 VHH antibody includes a single epitope tag (single tag sequence) or multiple tags (multiple tag sequences), to which anti-tag antibodies specifically bind. Such epitope tags, which are specifically bindable by the anti-epitope tag antibodies, are useful in detecting VHHs bound to CLDN18.2 or CLDN18.1 protein antigen. The epitope tags may be placed at the amino terminus, carboxy terminus, or internally within a VHH molecule. Such tags and / or anti-tag antibodies are described for example, in (U. S. Patent No. 8,349,326; 9,023,352, WO 2019 / 094095A1) and U. S. Patent Nos. 7,943,345; 8,114,634 and 8,865,871), the contents of which are incorporated herein by reference in their entireties. By way of illustrative example, peroxidase labeled antibodies that bind the anti-O-tag antibody may be used to detect these anti-tag antibodies in assays in which samples are incubated with goat anti-O-tag-HRP conjugated antibody (Bethyl labs) diluted 1:5000 in blocking solution for 1 hour at RT with rocking and were washed as above before adding TMB microwell peroxidase substrate (KPL) to develop (incubated for 10-40 min). Development was stopped with IM H2SO4 and the plates were read at 450nm on an ELx808 Ultra Microplate Reader (Bio-Tek instruments), (Mukherjee, J. et al., 2012, PloS ONE 7:e29941). Chimeric Antigen Receptors (CAR) and CAR-T cells

[0200] The disclosure provides immune cells that express chimeric antigen receptors (CARs) containing anti-CLDN18.2 or anti-CLDN18.1 VHH domains (e.g., CLDN 9.4, CLDN_11.05, CLDN11.11, CLDN_11.13). as antigen binding domains. Non-limiting examples of CARs include those described in Larson and Maus, “Recent advances and discoveries in the mechanisms and functions of CAR-T cells, ” Nature Reviews Cancer, 21:145-161 (2021), the disclosure of which is incorporated herein by reference in its entirety for all purposes. Modification of immune cells to express a chimeric antigen receptor can enhance an immune cell’s immunoreactive activity. In embodiments, the chimeric antigen receptor has an affinity for an epitope on CLDN18.2 or CLDN18.1. In some instances, the chimeric antigen receptor selectively binds CLDN18.2. In some cases, the antigen is associated with an altered fitness of an organism. For example, the chimeric antigen receptor can have an affinity for an epitope CLDN18.2 expressed in a neoplastic cell. Because the CAR-T cells can act independently of major histocompatibility complex (MHC), activated CAR-T cells can kill the neoplastic cell expressing the antigen.

[0201] Some embodiments of the methods provided herein involve autologous immune cell immunotherapy, wherein immune cells are obtained from a subject having a disease or altered fitness characterized by cancerous or otherwise altered cells expressing a surface marker. The obtained immune cells are genetically modified to express a chimeric antigen receptor and are effectively redirected against specific antigens. Thus, in some embodiments, immune cells are obtained from a subject in need of CAR-T immunotherapy. In some embodiments, these autologous immune cells are cultured and modified shortly after they are obtained from the subject. In other embodiments, the autologous cells are obtained and then stored for future use. This practice may be advisable for individuals who may be undergoing parallel treatment that will diminish immune cell counts in the future. In allogeneic immune cell immunotherapy, immune cells can be obtained from a donor other than the subject who will be receiving treatment. In some embodiments, immune cells are obtained from a healthy subject or donor and are genetically modified to express a chimeric antigen receptor and are effectively redirected against specific antigens. The immune cells, after modification to express a chimeric antigen receptor, are administered to a subject for treating a neoplasia (e.g., a cancer). In some embodiments, immune cells to be modified to express a chimeric antigen receptor can be obtained from pre-existing stock cultures of immune cells.

[0202] Immune cells and / or immune effector cells can be isolated or purified from a sample collected from a subject or a donor using standard techniques known in the art. For example, immune effector cells can be isolated or purified from a whole blood sample by lysing red blood cells and removing peripheral mononuclear blood cells by centrifugation. The immune effector cells can be further isolated or purified using a selective purification method that isolates the immune effector cells based on cell-specific markers such as CD25, CD3, CD4, CD8, CD28, CD45RA, or CD45RO. In one embodiment, CD4+is used as a marker to select T cells. In one embodiment, CD8+is used as a marker to select T cells. In one embodiment, CD4+and CD8+are used as a marker to select regulatory T cells.

[0203] One technique for isolating or purifying immune effector cells is flow cytometry. In fluorescence activated cell sorting a fluorescently labelled antibody with affinity for an immune effector cell marker is used to label immune effector cells in a sample. A gating strategy appropriate for the cells expressing the marker is used to segregate the cells. For example, T lymphocytes can be separated from other cells in a sample by using, for example, a fluorescently labeled antibody specific for an immune effector cell marker (e.g., CD4, CD8, CD28, CD45) and corresponding gating strategy. In one embodiment, a CD4 gating strategy is employed. In one embodiment, a CD8 gating strategy is employed. In one embodiment, a CD4 and CD8 gating strategy is employed. In some embodiments, a gating strategy for other markers specific to an immune effector cell is employed instead of, or in combination with, the CD4 and / or CD8 gating strategy.

[0204] The immune effector cells contemplated in the disclosure are effector T cells or N K cells. In some embodiments, the effector T cell is a naive CD8+T cell, a cytotoxic T cell, a natural killer T (N KT) cell, a natural killer cell, a gammadelta T cell (y 5 T cell), or a regulatory T (Treg) cell. In some embodiments, the effector T cells are thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments the immune effector cell is a CD4+CD8+T cell or a CD4⁻ CD8⁻ T cell. In some embodiments the immune effector cell is a T helper cell. In some embodiments the T helper cell is a T helper 1 (Thl), a T helper 2 (Th2) cell, or a helper T cell expressing CD4 (CD4+ T cell). In some embodiments, the immune effector cell is any other subset of T cells.

[0205] Chimeric antigen receptors (CARs) as contemplated in the present disclosure comprise an extracellular binding domain (e.g., a VHH domain), a transmembrane domain (e.g., a transmembrane domain derived from a CD28, CD8, or CD3 polypeptide), and an intracellular domain (e.g., an intracellular domain containing a CD3ζ signaling domain, and / or a co-stimulation domain derived from a CD28, 4 IBB, 0X40, or CD27 polypeptide). Binding of an antigen to the extracellular binding domain can activate the CAR-T cell and generate an effector response, which includes CAR-T cell proliferation, cytokine production, and other processes that lead to the death of the antigen expressing cell. In some embodiments of the present disclosure, the chimeric antigen receptor further comprises a linker (e.g., a hinge domain derived from a CD8 CD28, CD4, or IgG polypeptide). In some cases, the CAR comprises a signal peptide, such as a signal peptide derived from a CD28 or GCSF signal peptide.

[0206] In various embodiments, the CAR specifically binds any antigen that can be targeted by a chimeric antigen receptor (CAR), such as CLDN18.2 or CLDN18.1. Further nonlimiting examples of antigens that can be bound by a CAR of the present disclosure include those described in Xu, et al. “The development of CAR design for tumor CAR-T cell therapy, ” Oncotarget. 9(17) doi: 10.18632 / oncotarget.24179, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0207] Chimeric antigen receptors, or any polypeptide of the present disclosure, can be delivered to an immune cell using a polynucleotide encoding the chimeric antigen receptor or polypeptide. For example, immune cells obtained from a subject may be transduced with a nucleic acid vector encoding the chimeric antigen receptor. The vector may then be used to transduce recipient immune cells so that these cells will then express the chimeric antigen receptor. Efficient means of transducing immune cells include transfection and transduction. Such methods are well known in the art. For example, applicable methods for delivery the nucleic acid molecule encoding the chimeric antigen receptor (and the nucleic acid(s) encoding the base editor) can be found in International Application No. PCT / US2009 / 040040 and US Patent Nos. 8, 450,112; 9,132,153; and 9,669,058, each of which is incorporated herein in its entirety. Additionally, those methods and vectors described herein for delivering the nucleic acid encoding the base editor are applicable to delivering the nucleic acid encoding the chimeric antigen receptor.

[0208] Extracellular Binding Domain

[0209] The chimeric antigen receptors of the disclosure include an extracellular binding domain (e.g., an anti-CLDN18.2 or anti-CLDN18.2 VHH domain, such as CLDN 9.4, CLDN 11.05, CLDN11.11, CLDN 11.13). The extracellular binding domain of a chimeric antigen receptor contemplated herein comprises an amino acid sequence of an antibody (e.g., a VHH antibody), or an antigen binding fragment thereof, that has an affinity for CLDN18.2 or CLDN18.1. In some embodiments the chimeric antigen receptor contains an amino acid sequence of an antibody (e.g., an anti-CLDN18.2 or anti-CLDN18.1 VHH domain, such as CLDN 9.4, CLDN 11.05, CLDN11.11, CLDN 11.13). In some embodiments, the chimeric antigen receptor contains the amino acid sequence of an antigen binding fragment of an antibody. The antibody (or fragment thereof) portion of the extracellular binding domain recognizes and binds to an epitope of CLDN18.2 or CLDN18.1. In some embodiments, the antibody fragment portion of a chimeric antigen receptor is a VHH domain. In other embodiments, the antibody fragment portion of a chimeric antigen receptor is a multichain variable fragment, which can comprise more than one extracellular binding domains and therefore bind to more than one antigen simultaneously. In a multiple chain variable fragment embodiment, a hinge region may separate the different variable fragments, providing necessary spatial arrangement and flexibility.

[0210] In embodiments, the antigen-binding portion of a chimeric antigen receptor comprises complementarity determining regions (e.g., CDR1, CDR2, and CDR3 regions) that are responsible for the antibody’s affinity for a particular antigen. Thus, antibodies that recognize different antigens comprise different complementarity determining regions.

[0211] In some embodiments, the antigen recognized and bound by the extracellular domain is a protein or peptide, a nucleic acid, a lipid, or a polysaccharide. Antigens can be heterologous, such as those expressed in a pathogenic bacteria or virus. Antigens can also be synthetic; for example, some individuals have extreme allergies to synthetic latex and exposure to this antigen can result in an extreme immune reaction. In some embodiments, the antigen is autologous, and is expressed on a diseased or otherwise altered cell. For example, in some embodiments, the antigen is expressed in a neoplastic cell.

[0212] Transmembrane Domain

[0213] The chimeric antigen receptors of the disclosure include a transmembrane domain. The transmembrane domain of the chimeric antigen receptors described herein spans the CAR-T cell’s or CAR NT cell’s lipid bilayer cellular membrane and separates the extracellular binding domain and the intracellular signaling domain. In some embodiments, this domain is derived from other receptors having a transmembrane domain, while in other embodiments, this domain is synthetic. In some embodiments, the transmembrane domain may be derived from a non-human transmembrane domain and, in some embodiments, humanized. By “humanized” is meant having the sequence of the nucleic acid encoding the transmembrane domain optimized such that it is more reliably or efficiently expressed in a human subject. In some embodiments, the transmembrane domain is derived from another transmembrane protein expressed in a human immune effector cell.

[0214] Intracellular Signaling Domain

[0215] The chimeric antigen receptors of the disclosure include an intracellular signaling domain. The intracellular signaling domain is the intracellular portion of a protein expressed in a T cell or N K cell that transduces an effector function signal ( e.g., an activation signal) and directs the effector cell to perform a specialized function. Effector cell activation can be induced by a number of factors, including binding of cognate antigen to the chimeric antigen receptor on the surface of effector cell and / or binding of cognate ligand to costimulatory molecules on the surface of the cell. An effector cell co-stimulatory molecule is a cognate binding partner on an immune effector cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the effector cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, an MHC class I molecule. Activation of an effector cell leads to immune response, such as effector cell proliferation and differentiation (see, e.g., Smith-Garvin etal., Annu. Rev. Immunol., 27:591-619, 2009). Exemplary effector cell (e.g., T cells) signaling domains are known in the art.

[0216] The intracellular signaling domain of the chimeric antigen receptor contemplated herein comprises a primary signaling domain. In some embodiments, the chimeric antigen receptor comprises the primary signaling domain and a secondary, or co-stimulatory, signaling domain.

[0217] Polynucleotides and vectors

[0218] In some cases, more than one anti-CLDN18.2 or anti-CLDN18.1 VHH antibody (i.e., anti-CLDN18.2 or anti-CLDN18.1 VHH) is coupled or linked (e.g., covalently linked) to other sequences, e.g., a leader amino acid sequence, domains of a chimeric antigen receptor, one or more spacer or linker ( flexible spacer or linker) amino acid sequences, or one or more epitope tag amino acid sequences. In an embodiment, a polynucleotide molecule, such as a recombinant or isolated polynucleotide molecule, encodes a polypeptide containing an anti-CLDN18.2 or anti-CLDN18.1 VHH polypeptide (e.g., a VHH antibody or a chimeric antigen receptor). In an embodiment, the polynucleotide encodes a fragment or portion of the anti-CLDN18.2 or anti-CLDN18.1 VHH, where the fragment or portion maintains CLDN18.2 or CLDN18.1 binding activity. In an embodiment, an VHH antibody may be humanized, i.e., modified to increase its similarity to antibodies or antibody variants produced naturally in humans, using techniques known and practiced in the art. Briefly and by way of nonlimiting example, a humanized antibody can be generated by inserting the appropriate CDR coding sequences into a human antibody scaffold comprising essentially invariant framework region (FR) sequences (FRs). In embodiments, the CDRs of the anti-CLDN18.2 or anti-CLDN18.2 VHH antibodies described herein may be inserted into FRs, which provide the structural scaffold that allows the CDRs to bind to CLDN 18.2 or CLDN 18.1. Recombinant DNA methods using an appropriate vector and expression in mammalian cells are employed and routinely practiced in the art to achieve the production of recombinant humanized antibodies.

[0219] In an embodiment, the polynucleotide encodes a CLDN18.2 or CLDN18.1 binding VHH molecule having binding function, or a functional binding portion thereof. In embodiments, antibody fragments, microproteins, designed ankyrin repeat proteins (DARPins), anticalins, peptide mimetic molecules, aptamers, synthetic molecules, etc. can be linked to the anti-CLDN18.2 or anti-CLDN18.1 V HH binding molecule.

[0220] In an embodiment, an anti-CLDN18.2 or anti-CLDN18.1 VHH can be modified, for example, by attachment (e.g., either directly or indirectly via a linker or spacer) to another agent (e.g., a detectable label, a cytotoxic drug, and / or another polypeptide). Accordingly, a polynucleotide (e.g., DNA) that encodes one anti-CLDN18.2 V HH or anti-CLDN18.1 VHH is joined (in reading frame) with a polynucleotide encoding a second polypeptide, and so on. In certain embodiments, additional amino acids are encoded within the polynucleotide between the anti-CLDN18.2 or anti-CLDN18.1 VHH and other polypeptides so as to produce an unstructured region (e.g., a flexible spacer) that separates the anti-CLDN18.2 or anti-CLDN18.1 VHH from the other polypeptides to better promote independent folding of each polypeptide into its active or functional conformation or shape. Commercially available techniques for fusing proteins (or their encoding polynucleotides) may be employed to recombinantly join or couple polypeptide sequences to one another.

[0221] The compositions and methods described herein in various embodiments include an isolated polynucleotide sequence or an isolated polynucleotide molecule that encodes a polypeptide (e.g., anti-CLDN18.2 VHH, anti-CLDN18.1 VHH, or a chimeric antigen receptor containing an anti-CLDN18.2 VHH or anti-CLDN18.1 VHH domain of the disclosure). Accordingly, in some embodiments, the isolated polynucleotide sequence or isolated polynucleotide molecule comprises or consists of a polynucleotide sequence that encodes a polypeptide molecule (anti-CLDN18.2 V HH or anti-CLDN18.1 VHH) having an amino acid sequence listed in Table 1 or 8, or a functional portion thereof, as described herein. In an embodiment, a composition comprises a combination of the isolated polynucleotide sequences or isolated polynucleotide molecules.

[0222] Also encompassed by the present disclosure are polynucleotide sequences, DNA or RNA, which are substantially complementary to the DNA sequences encoding the polypeptides described herein, and which specifically hybridize with these DNA sequences under conditions of stringency known to those of skill in the art. As referred to herein, substantially complementary means that the nucleotide sequence of the polynucleotide need not reflect the exact sequence of the original encoding sequences, but must be sufficiently similar in sequence to permit hybridization with a nucleic acid sequence under high stringency conditions. For example, non-complementary bases can be interspersed in a nucleotide sequence, or the sequences can be longer or shorter than the polynucleotide sequence, provided that the sequence has a sufficient number of bases complementary to the sequence to allow hybridization thereto. Conditions for stringency are described, e.g., in Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994), and Brown, et al., Nature, 366:575 (1993); and further defined in conjunction with certain assays.

[0223] Vectors and plasmids containing one or more of the polynucleotide molecules encoding the anti-CLDN18.2 VHH amino acid sequences of Table 1 or 8, or a functional portion thereof, are provided. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by the skilled practitioner in the art. Additional vectors can also be found, for example, in Ausubel, F. M., et al., Ibid, and in Sambrook et al”, “Molecular Cloning: A Laboratory Manu’T, 2nd ED. (1989), and other editions.

[0224] Uses of plasmids, vectors or viruses (viral vectors) containing polynucleotides encoding the anti-CLDN18.2 VHHs as described herein include generation of mRNA or protein in vitro or in vivo. In related embodiments, host cells transformed with the plasmids, vectors, or virus vectors are provided, as described above. Nucleic acid molecules can be inserted into a construct (such as a prokaryotic expression plasmid, a eukaryotic expression vector, or a viral vector construct, which can, optionally, replicate and / or integrate into a recombinant host cell by known methods. The host cell can be a eukaryote or prokaryote and can include, for example and without limitation, yeast (such as Pichia pastoris or Saccharomyces cerevisiae), bacteria (such as E. coli, or Bacillus subtilis), animal cells or tissue (CHO or COS cells), insect Sf9 cells (such as baculoviruses infected SF9 cells), or mammalian cells (somatic or embryonic cells, Human Embryonic Kidney (HEK) cells, Chinese hamster ovary (CHO) cells, HeLa cells, human 293 cells (Expi293F), and monkey COS-7 cells). Suitable host cells also include a mammalian cell, a bacterial cell, a yeast cell, an insect cell, a plant cell, or an algal cell.

[0225] In another aspect, an RNA polynucleotide, in particular, mRNA, encodes a polypeptide as described herein. mRNA encoding the polypeptides may contain a 5' cap structure, a 5' UTR, an open reading frame, a 3' UTR and poly-A sequence followed by a C30 stretch and a histone stem loop sequence (Thess, A. et al., 2015, Mol Ther, 23(9): 1456-1464; Thran, M. et al., 2017, EMBO Molecular Medicine, DOI:

[0226] 10.15252 / emmm.201707678). Sequences may be codon-optimized for human use using techniques and protocols known and used by those skilled in the art. In an embodiment, the mRNA sequences do not include chemically modified bases. mRNAs encoding the anti-CLDN18.2 VHHs thereof as described herein may be capped enzymatically or further polyadenylated for in vivo studies / use. In an embodiment, a polypeptide of the disclosure is encoded by a mRNA molecule. In an embodiment, the mRNA may be delivered to or introduced into a cell.

[0227] Expression of proteins, which normally have a shortened serum half-life, by encoding mRNA, particularly sequence optimized, unmodified mRNA, advantageously prolongs the bioavailability of these proteins for in vivo activity, (see, e.g., K. Kariko et al, 2012, Mol. Ther., 20:948-953; Thess, A. et al., 2015, Mol Ther, 23(9): 1456-1464;).

[0228] Recombinant Polypeptide Expression

[0229] In general, polypeptides of the disclosure (e.g., VHH antibodies) may be produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle.

[0230] Those skilled in the field of molecular biology will understand that any of a wide variety of systems may be used to express a recombinant protein. The precise host cell used is not critical to the various aspects and embodiments of the disclosure. A polypeptide of the disclosure may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., an immune cell, such an immune effector cell (e.g., a T cell) Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., supra). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 198 7).

[0231] A variety of expression systems exist for the production of the polypeptides (e.g., VHH antibodies or chimeric antigen receptors) of the disclosure. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.

[0232] One particular bacterial expression system for polypeptide production is the E. coli pET expression system ( Novagen, Inc., Madison, Wis). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.

[0233] Another bacterial expression system for polypeptide production is the pGEX expression system ( Pharmacia). This system employs a GST gene fusion system that is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.

[0234] Once the recombinant polypeptide of the disclosure is expressed, it can be isolated, e.g., using affinity chromatography. In one example, an antibody (e.g., produced as described herein) raised against an antigen of the disclosure may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods.

[0235] Once isolated, a recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides of the disclosure, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis’ 2nd ed., 198 4 The Pierce Chemical Co., Rockford, Ill.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).

[0236] Compositions

[0237] Provided also are compositions (e.g., pharmaceutical compositions) for use in the methods of the disclosure. In embodiments, the composition is a pharmaceutical composition for use in treating a disease or disorder (e.g., a neoplasia). In some instances, a composition of the disclosure is used in a diagnostic method (e.g., to detect a marker associated with a disease). In some cases, a composition of the disclosure is used in one of the screening methods provided herein. In an embodiment, the compositions contain a cell, polynucleotide, vector, or polypeptide (e.g., a VHH antibody) provided herein. In some cases, the composition contains CAR-T cells or CARNK cells, as described herein and an acceptable carrier, excipient, or diluent.

[0238] The agents of the disclosure (e.g., polynucleotides, polypeptides, vectors, and / or cells) may be contained in any appropriate amount in any suitable carrier substance and is / are present in some cases in an amount of 0.01-95% by weight of the total weight of the composition. A pharmaceutical composition may be provided in a form that is suitable for a parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration route, such that the agent, such as a vector or cell described herein, is systemically delivered.

[0239] The compositions of the present disclosure can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (1st ed. 2005). In some embodiments, an agent of the disclosure is present in a reconstitutable dry composition (e.g., a lyophilized composition or powder). In embodiments, an agent is admixed with a suitable carrier prior to administration or storage, and in some embodiments, the composition further comprises an acceptable carrier (e.g., a pharmaceutically acceptable carrier). Suitable pharmaceutically acceptable carriers generally comprise inert substances that aid in administering the pharmaceutical composition to a subject, aid in processing the pharmaceutical compositions into deliverable preparations, or aid in storing the pharmaceutical composition prior to administration. Carriers can include agents that can stabilize, optimize or otherwise alter the form, consistency, viscosity, pH, pharmacokinetics, or solubility of a composition. Such agents include buffering agents, wetting agents, emulsifying agents, diluents, encapsulating agents, and skin penetration enhancers. For example, carriers can include, but are not limited to, saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.

[0240] Some nonlimiting examples of materials which can serve as carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Rin’er's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.

[0241] Compositions of the disclosure can contain one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine. Alternatively, the pH buffering compound is an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions.

[0242] Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions. The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level.

[0243] Compositions can also contain one or more osmotic modulating agents, z.e., a compound that modulates the osmotic properties (e.g., tonicity, osmolality, and / or osmotic pressure) of the formulation to a level that is acceptable, for example, to the blood stream and blood cells of recipient subjects. The osmotic modulating agent can be an agent that does not chelate calcium ions. The osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation. Illustrative examples of suitable types of osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars or sugar alcohols, such as sucrose, pentose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and / or types of agents. The osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation.

[0244] The skilled artisan can readily determine the number of cells and amount of optional additives, vehicles, and / or carriers in compositions and to be administered in methods of the disclosure. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is advantageous to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model (e.g., a rodent such as a mouse); and, the dosage of the composition(s), concentration of components therein, and the timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein, and the time for sequential administrations can be ascertained without undue experimentation.

[0245] In some embodiments, the composition is formulated for delivery to a subject.

[0246] Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intracerebroventricular administration. The pharmaceutical composition may be administered systemically.

[0247] The composition may be in the form of a solution or suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the agent (e.g., CAR-T cells, CARNK cells, VHH antibodies, polynucleotides, or polypeptides provided herein), the composition may include suitable parenterally acceptable carriers and / or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and / or dispersing, agents.

[0248] In some embodiments, the composition is formulated for intravenous delivery. The compositions according to the described embodiments may be in a form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that may be employed include water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1, 3 -butanediol, Rin’er's solution, isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w / w of propylene glycol or the like.

[0249] Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as chickens, ducks, geese, and / or turkeys.

[0250] Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the composition, its use is contemplated to be within the scope of this disclosure.

[0251] In some embodiments, compositions in accordance with the present disclosure can be used for treatment of any of a variety of diseases, disorders, and / or conditions. Treatments

[0252] The compositions, polynucleotides, cells (e.g., chimeric antigen receptor T or N K cells), and / or polypeptides (e.g., polypeptides containing a VHH antibody of the disclosure, or a functional fragment thereof) provided herein can be used for treating a subject for a disease or disorder, such as a neoplasia (e.g., a lung cancer or an ovarian cancer). Generally, the methods provided herein include administering a therapeutically effective amount of an agent as provided herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.

[0253] The methods provided herein include selecting a subject for and / or administering to a subject having or having a propensity to develop a neoplasia a treatment that includes a therapeutically effective amount of an immunotherapeutic agent, such as a CAR-T cell or CAR N K cell of the disclosure.

[0254] In some instances, the methods provided herein involve administering to a subject in need thereof a VHH domain provided herein that has been conjugated to an agent (e.g., a cytotoxic agent). Antibody-drug conjugates are described, e.g., in Zolog, et al. “Antibodydrug conjugates, ” Nature Reviews Drug Discovery, 12:259-260 (2013), the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0255] An effective amount of an agent can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound or agent (i.e., an effective dosage) depends on the therapeutic compounds or agents selected. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic agents provided herein can include a single treatment or a series of treatments.

[0256] Dosage, toxicity and therapeutic efficacy of the therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Agents which exhibit high therapeutic indices may be used. While agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0257] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents lies in various embodiments within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses more accurately in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0258] Dosages and desired drug concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration (e.g., oral administration, intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intracranial, intraspinal, subcutaneous, intraarticular, intrasynovial, intrathecal, topical, or inhalation routes) is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles described in Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics, ” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.

[0259] For in vivo administration of any of the agents of the present disclosure, normal dosage amounts may vary from about 10 ng / kg up to about 100 mg / kg of an individual's and / or subject's body weight or more per day, depending upon the route of administration. In some embodiments, the dose amount is about 1 mg / kg / day to 10 mg / kg / day. In some embodiments, the dose amount of a CAR-T cell is about, at least about, and / or no more than about 1e5 cells, 1e6 cells, 1e7 cells, 1e8 cells, 1e9 cells, 1e10 cells, 1e11 cells, 1e12 cells, 1e13 cells, 1e14 cells, 1e15 cells, or 1e16 cells. For repeated administrations over several days or longer, depending on the severity of the disease, disorder, or condition to be treated, the treatment is sustained until a desired suppression of symptoms is achieved. An effective amount of an agent of the instant disclosure may vary, e.g., from about 0.001 mg / kg to about 1000 mg / kg or more in one or more dose administrations for one or several days (depending on the mode of administration). In certain embodiments, the effective amount per dose varies from about 0.001 mg / kg to about 1000 mg / kg, from about 0.01 mg / kg to about 750 mg / kg, from about 0.1 mg / kg to about 500 mg / kg, from about 1.0 mg / kg to about 250 mg / kg, and from about 10.0 mg / kg to about 150 mg / kg.

[0260] An exemplary dosing regimen may include administering an initial dose of an agent of the disclosure of about 200 pg / kg, followed by a weekly maintenance dose of about 100 pg / kg every other week. Other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. For example, dosing an individual from one to twenty-one times a week is contemplated herein. In certain embodiments, dosing ranging from about 3 pg / kg to about 2 mg / kg (such as about 3 pg / kg, about 10 pg / kg, about 30 pg / kg. about 100 pg / kg, about 300 pg / kg, about 1 mg / kg. or about 2 mg / kg) may be used. In certain embodiments, dosing frequency is three times per day, twice per day, once per day. once every other day. once weekly, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once monthly, once every two months, once every three months, or longer. Progress of the therapy is easily monitored by conventional techniques and assays. The dosing regimen, including the agent(s) administered, can vary over time independently of the dose used.

[0261] Methods for characterizing the efficacy of a treatment for a neoplasia are well known in the art (e.g., computerized tomography (CT) scan, bone scan, magnetic resonance imaging (MRI), positron emission tomography ( PET) scan, ultrasound X-ray, biopsy, etc.).

[0262] Substantially identical amino acid and nucleotide sequences for VHHs

[0263] There is a large body of information in the literature supporting the fact that closely related antibody (Ab) sequences are capable of performing the same binding and therapeutic functions such that this is now generally accepted by those with ordinary skill in the art of immunological sciences. The creation of Abs with small numbers of amino acid sequence variations occurs naturally within mammals and some other animal species during the process of ‘affinity maturation’ in which Ab-producing cells that bind a newly encountered antigen (Ag) are expanded, and their progeny cells contain random mutations within portions of the Ab coding DNA that results in new, related Ab sequences. The cells expressing Abs that have gained improved binding properties for the new Ag are then selected and expanded, thereby increasing the amount of the improved antibody in the animal. This process continues through multiple generations of mutation and selection until Abs with greatly improved antigen binding properties result. The process of Ab affinity maturation demonstrates that related, yet not identical, Ab amino acid sequences can possess similar target binding properties and perform similar therapeutic functions in vivo.

[0264] The present disclosure provides anti-CLDN18.2 and anti-CLDN18.1 VHH antibodies having related sequences that are capable of binding CLDN18.2 and / or CLDN18.1. The Abs described herein are heavy-chain only, single domain VHH antibodies, which are generated in camelid alpacas, which have been reported to be convenient sources of camelid VHH antibodies (See, e.g., Maass, D. R. et al., 2007, J. Immunol. Methods, 324:13-25). Briefly, an animal capable of producing VHH antibodies in response to an antigen are immunized with a selected CLDN18.2 or CLDN18.1 antigen (CLDN18.2 Ag or CLDN18.1 Ag) one or multiple times to permit the animal to undergo affinity maturation of the anti-CLDN18.2 or anti-CLDN 18.1 V HHs that are produced. Anti-CLDN 18.2 and anti-CLDN 18.1 V HHs are then isolated and the encoding DNA selected for expression of soluble V HHs that bind CLDN18.2 Ag or CLDN18.1 Ag and have potential therapeutic or diagnostic properties. During this process, many examples of closely related anti-CLDN18.2 or anti-CLDN18.1 binding V HHs are isolated, which are distinctive, and which are presumably intermediates that result from the affinity maturation process which occurs during anti-CLDN18.2 or anti-CLDN18.1 VHH production in alpaca lymphocytes. These related anti-CLDN18.2 VHHs or anti-CLDN18.1 VHHs are screened for binding to CLDN18.2 or CLDN18.1 Ag, and the most promising members of homology groups of CLDN18.2 or CLDN18.1 binding V HHs are identified and become lead candidates for further development.

[0265] Similar to all mammalian antibodies, VHHs consist of four, well-conserved ‘framework’ regions (FRs) which are important in forming the antibody structure. Between the FRs (FR1, FR2, FR3 and FR4) are three much less well-conserved CDRs or hypervariable regions (CDR1, CDR2 and CDR3) which principally interact with and bind to antigenic determinants or epitopes on antigens (Ags), such as CLDN18.2 or CLDN18.1. The CDR sequences vary widely so as to interact and bind to epitopes of Ags. The third CDR, CDR3, is generally the longest in sequence and is most diverse of the CDRs within VHHs, both in size and sequence. By way of nonlimiting example, CDR3 in VHHs can range in size from about 5 to about 30 amino acid residues. Without intending to be bound by theory, VHHs and CDR3 regions that bind to the same CLDN18.2 or CLDN18.1 target Ag are considered to have resulted from affinity maturation of a common precursor V HH within the animal and are classified as a ‘homology group.’ Individual VHHs within a homology group are classified by their binding to the target Ag, and the members of the VHH homology group are able to ‘compete’ with each other for binding to the Ag, thus demonstrating that they bind to the same region on the target Ag. In VHH molecules, the CDRs (CDR1, CDR2 and CD3) play a role in the ability of a VHH to bind to the target Ag, e.g., CLDN18.2 or CLDN18.1, in conjunction with CDR1 and CDR2.

[0266] Since the FRs maintain the structure of a V HH and the positioning of the CDRs for binding to the target Ag, the FRs of V HHs typically do not vary extensively in sequence. However, some VHH FR amino acid sequence variation is permissible, particularly in cases in which an amino acid substitution involves the replacement or substitution of one amino acid with another amino acid having similar properties (e.g., similarity in being charged or uncharged), i.e., a conservative substitution. Such conservative changes in FRs can often be found naturally within VHHs that have undergone affinity maturation in an animal. Similar to the case with FRs, VHH CDRs also typically do not vary extensively in amino acid sequence or type so as not to compromise their ability to specifically bind to Ag. As would be appreciated by one skilled in the art, an estimation of the extent of amino acid sequence variation that can be tolerated within V HHs without compromising their Ag binding ability can be made by observing the variation that occurs naturally within affinity-matured homology groups of V HHs isolated from the same types of animals and which bind to the same Ag.

[0267] In an embodiment, sequence variation is particularly acceptable in the CDR regions, e.g., CDR1, CDR2, and / or CDR3, while the feature of V HH binding to antigen is maintained. In an embodiment, amino acid sequence variation results from conservative amino acid substitutions in a VHH sequence. In an embodiment, the conservative amino acid substitutions are in one or more CDR sequences of the VHH polypeptide. In an embodiment, the conservative amino acid substitutions are in one or more FR sequences of the VHH polypeptide. In an embodiment, the conservative amino acid substitutions are in one or more CDR sequences and in one or more FR sequences of the VHH polypeptide.

[0268] An example evidencing that V HH sequence variation is acceptable within related VHHs having the same Ag binding characteristics is described in Tremblay et al., 2013, Infect Immun 81:4592-4603. In this report, 11 V HH sequences comprise a large homology group with closely related CDR3 sequences, and the unusual property of cross-specific binding to two different Shiga toxins, Stxl and Stx2. Two of the more distantly related VHH members of this homology group are characterized as having common Ag binding characteristics. These two related VHHs were found to have 32 amino acid changes in the total VHH sequence of 120 or 121 residues. Thus, a 26% variation in amino acid sequence did not adversely affect the common Ag binding properties of the VHH proteins.

[0269] Kits

[0270] The disclosure also provides kits for use in the methods of the disclosure. Kits of the instant disclosure may include one or more containers comprising an agent provided herein, such as a polypeptide (e.g., a VHH antibody) or a polynucleotide encoding the same or a cell (e.g., a CAR-T cell). In some embodiments, the kits further include instructions for use in accordance with the methods of this disclosure. In some embodiments, these instructions comprise a description of use of the agent to treat a disease or identify VHH antibodies that bind a target antigen. The kit may further comprise a description of how to analyze and / or interpret data.

[0271] Instructions supplied in the kits of the instant disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions may be provided for practicing any of the methods described herein.

[0272] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

[0273] The practice of the various aspects and embodiments of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” ( Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);

[0274] “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the various aspects and embodiments of the disclosure. Particularly useful techniques for specific embodiments will be discussed in the sections that follow.

[0275] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention.

[0276] EXAMPLES

[0277] Example 1: Discovery of anti-CLDN18.2 VHH antibodies

[0278] Experiments were undertaken to develop VHH antibodies capable of binding CLDN18.2. To develop the VHH antibodies, mice (i.e., nanomice) capable of producing VHH antibodies (i.e., nanobodies) in response to an antigen were vaccinated in two separate rounds with a live vaccine containing antigen presenting cells expressing CLDN18.2 (e.g., as shown in FIG. 1A). The antigen presenting cells were DC2.4 cells transduced with expression constructs encoding CLDN18.2 (FIG. IB). The transduced DC2.4 cells surface-presented CLDN18.2 antigens. Nanomice were initially vaccinated with 5E7 CLDN18.2+ DC2.4 cells intraperitoneally (IP) with 20 pg of CpG adjuvant. Mice were then given two follow up boost vaccinations at 14 days and 28 days, each again with 5E7 CLDN18.2+ DC2.4 cells IP with 20 pg of CpG adjuvant. Blood was collected 3-4 days after each vaccination boost and serum was isolated. Serum was used as a polyclonal antibody mix to immunolabel LentiX cells transgenically expressing CLDN18.1, CLDN18.2, or a mismatched antigen control, mesothelin (MSLN), followed by immunolabeling with AF647 antimouse Fc secondary antibody ( FIG. 1C). Evaluation of serum samples from the vaccinated nanomice confirmed that the mice produced anti-CLDN18.2 VHH antibodies, and were negative for anti-mesothelin antibodies ( FIG. ID). As expected, CLDN18.1+ cells were effectively labeled using serum from mice vaccinated using the CLDN18.2+ cells to a lesser extent, given that the first extracellular loop of the two isoforms are 85% similar by amino acid sequence.

[0279] Having established that the vaccinated nanomice produced anti-CLDN18.2 nanobodies, experiments were undertaken to generate nanobody repertoire libraries ( “vaccination libraries”) for subsequent experimentation. First, cDNA was prepared using nanobody-encoding mRNA from the nanomice and the encoded nanobodies were cloned inframe into an expression vector encoding a chimeric antigen receptor (CAR) such that the nanobodies would function as the antigen-binding domain of the encoded CAR. The chimeric antigen receptors contained from N-terminus to C-terminus a CD28 signal peptide, a cloned VHH domain as an antigen-binding domain (ABD), a CD8 hinge domain, a CD28 transmembrane domain, a CD28 cytoplasmic domain, and a CD3( ^ domain. The expression construct further encoded an enhanced GF P (eGF P) protein linked to the C-terminus of the CAR by a P2A self-cleaving peptide. The expression vectors were cloned into bacteria and colonies containing the expression vectors were randomly selected and the amino acid sequences of VHH domains of the expression vectors corresponding to each colony were determined by next generation sequencing.

[0280] To identify maximally diverse anti-CLDN18.2 VHH antibody sequences, the full-length amino acid sequences were clustered and projected into t-SNE space (FIG. 2).

[0281] Clustering was performed by analyzing the amino acid relationships using a Cluster-K Spaced Amino Acid algorithm to determine similarity / dissimilarity between each sequence. The sequence clonotype abundance threshold was set to >5. The resulting relationship feature matrix was dimensionally reduced to 2D space by t-SNE then further cluster architecture was established by using K-means nearest neighbor. In addition, sequences from the anti-CLDN18.2 VHH antibodies CLDN_9.4, CLDN_11,13, CLDN_11.05, and CLDN_11.11 were mapped onto the data (see stars in FIG. 2). The plot revealed three distinct clusters to which the anti-CLDN18.2 VHH antibodies belonged. The sequences for all polypeptides falling within a cluster containing the CLDN_9.4, CLDN_11,13, CLDN_11.05, or CLDN l 1.11 antibodies are listed in Tables 1-3. The sequences within each cluster were highly similar and may represent additional CLDNl 8.1 and CLDNl 8.2 binders. The sequences of all related sequences within a cluster containing CLDN 9.4, CLDN l 1.13, CLDN l 1.05, and CLDN l 1.11 were then aligned using a multiple sequence alignment algorithm and are provided in Tables 6B, 6C, and 6K. Consensus sequences for complementarity determining regions corresponding to each cluster are provided in Table 7.

[0282] Example 2: In vitro validation of anti-CLDN18.1 / CLDN18.2 binders

[0283] Experiments were undertaken to validate the ability of anti-CLDN18.1 and anti-CLDN18.2 VHH antibodies identified in Example 1 to function as antigen-binding domains of chimeric antigen receptors expressed by T cells. Lentiviral vectors containing polynucleotides encoding the chimeric antigen receptors (CAR) containing anti-CLDN18.1 or anti-CLDN18.2 V HH domains as their antigen-binding domains were prepared and transduced into Jurkat cells containing an activation reporter cassette that expressed the fluorescent protein mKate when the cells expressing the CARs were activated by the CARs binding CLDN18.1 and CLDN18.2. Transduction efficiency was checked by flow cytometry to ensure suff icient coverage was maintained and that multiplicity of infection (MOI) was ~1. Transduced cells were co-cultured for 48 hours with blue fluorescent protein (BF P) expressing K562 cells that also transgenically expressed either CLDN18.2, CLDN18.1, or MUC16. Activation of Jurkats was assessed by flow cytometry (FIG. 3 A). Cells expressing vaccination library 9 demonstrated robust activation and CLDN18.2 specificity, and activation of cells expressing vaccination library 11 was approximately equal between CLDN 18.1 and CLDN 18.2. An experiment was undertaken to screen nanobodies from vaccination library 9 according to the method described in FIG. 7. Activated human T cells (human primary T cells) were transduced to express chimeric antigen receptors containing as their respective antigen binding domains VHH domains from vaccination library 9 to yield CAR T cells. The CAR T cells were expanded and sorted to purity using flow cytometry. The sorted cells were subsequently exposed to one of the following conditions: 2 rounds of stimulation with target cells expressing CLDN18.2, CLDN18.1, or neither CLDN18.1 nor CLDN18.2. As a control, the CAR T cells were grown in the absence of any target cells. The CAR T cells were subsequently sorted into CD25+ and CD25- fractions representing activated and non-activated cells, respectively, and the VHH domains corresponding to the chimeric antigen receptors represented in each fraction were then sequenced and analyzed. It was found that many of the CAR T cells showed specificity for activation by target cells expressing CLDN18.2 and that the number of cells showing activation increased with stimulations by the target cells ( FIG. 8). Libraries from each screen condition were successfully recovered ( FIG. 9). The screens identified several VHH domain sequences enriched in CAR T cells showing activation when contacted with target cells expressing CLDN18.2 ( FIG. 10). The V HH domain sequences were clustered using t-distributed stochastic neighbor embedding (t-SN E) ( FIGs. 11, 12, 13 A, and 13B) and identified a cluster of sequences containing the CLDN 9.4 VHH antibody domain sequence as containing anti-CLDN18.2 VHH antibody domain sequences. CDR3 sequences corresponding to this identified cluster of sequences are provided in FIG. 12.

[0284] Next, nanobodies from vaccination libraries 9 and 11 were further characterized ( FIG. 3B) Lentiviral vectors containing polynucleotides encoding chimeric antigen receptors (CAR) containing the selected anti-CLDN18.1 or anti-CLDN18.2 VHH domains as antigen-binding domains were prepared and transduced into Jurkat cells containing an activation reporter cassette that expressed the fluorescent protein mKate when the cells expressing the CARs were activated by the CARs binding CLDN18.1 and CLDN18.2. The Jurkat cells expressing the CARs were co-cultured with target K562 cell lines transgenically expressing CLDN18.2, CLDN18.1, or a mis-matched antigen to assess the activation potential of each CAR. The activation potential of more than 40 CARs from vaccination libraries 9 and 11 was measured by detecting expression of GF P (to measure CAR expression) and mKate (to measure cell activation) ( FIGS. 3C-D). As controls, the CAR-T Jurkat cells were also cultured in the presence of cells that did not express CLDN18.1, CLDN18.2, or in the absence of any other cells (i.e., CAR-Ts only). A highly selective CLDN18.2 binder in vaccination library 9 was discovered (referred to as “9.4 ”), and three pan CLDN18.1 / 18.2 binders were discovered ( “11.5”, “11.11”, and “11.13”).

[0285] Next, experiments were undertaken to demonstrate that T cells expressing chimeric antigen receptors containing 9.4, 11.5, 11.11, or 11.13 as an antigen binding domain (9.4 CAR-T cells, 11.5 CAR-T cells, 11.11 CAR-T cells, and 11.13 CAR-T cells, respectively) facilitated killing of target cells. Following a 48 hour in vitro co-culture assay, it was found that the 9.4 CAR-T cells were able to selectively control CLDN18.2+ K562 cells in an isoform / antigen specific fashion ( FIG. 3E). It was also found that the 11.5 CAR-T cells, 11.11 CAR-T cells, and 11.13 CAR-T cells were able to control K562 target cells expressing either the CLDN 18.1 or CLDN 18.2 isoform.

[0286] Experiments were undertaken to characterize the ability of the 9.4 VHH antibodies to selectively bind CLDN18.2. 6x His tagged 9.4 VHH Fc fusion antibodies were produced by Genscript in a mammalian cell line and purified. The purified 9.4 VHH antibodies were characterized using a flow cytometry -based assay to see how well they bound CLDN18.2 expressed on cells ( FIG. 4). K562 cells modified to surface-express CLDN18.1, CLDN18.2, or a mis-matched antigen control were immunostained using the purified nanobodies at concentrations ranging from <0.001 pg / mL to l. Opg / mL. Immunolabeling of the K562 cells with the Genscript synthesized 9.4 VHH Fc fusion antibodies demonstrated that the 9.4 VHH antibodies selectively bound CLDN18.2+ cells.

[0287] Experiments were undertaken to determine the impact of mutating CLDN18.2 on binding of the 9.4 VHH antibodies to CLDN18.2. Various alterations of amino acids or combinations of amino acids of CLDN18.2 to the corresponding amino acids in CLDN18.1 were made so that CLDN18.2 would more closely resemble CLDN18.1, and binding of the 9.4 VHH antibodies to the altered CLDN18.2 polypeptide was then evaluated. It was determined that no one amino acid alteration or combination of up to 3 amino acid alterations abrogated binding of the 9.4 VHH antibodies to CLDN18.2. In contrast to this finding, it was determined that any one of the amino acid alterations evaluated eliminated binding of a commercially available antigen-binding domain hu8E5 (see, e.g., Jiang, etal., J. Natl Cancer Inst., 111:409-418 (2019)) to CLDN18.2.

[0288] Ex ample 3: Mediation of killing of tumor cel ls in vivo by chimeric antigen receptors containing CLDN 9.4 as an antigen-binding domain

[0289] Experiments were undertaken to demonstrate that chimeric antigen receptors (CARs) containing as their antigen-binding domain an anti-CLDN18.2 VHH domain identified in the above examples mediated killing of target cells. Mice containing a GSU stomach adenocarcinoma were administered human primary T cells expressing a chimeric antigen receptor containing the VHH domain CLDN 9.4 as an antigen-binding domain (9.4-VHH CAR T cells). The 9.4-VHH CAR T cells were capable of killing the tumor cells in the mice ( FIG. 14) It was also found that the 9.4-V HH CAR T cells mediated killing of the tumor cells at levels comparable to T cells expressing a similar chimeric antigen receptor containing as its antigen binding domain the commercially available antigen-binding domain hu8E5 (see, e.g., Jiang, etal., J. Natl Cancer Inst., 111:409-418 (2019)).

[0290] The following materials and methods were employed in the above examples.

[0291] In vitro screening of VHH domains using prima ry human T cel ls

[0292] An unbiased in vitro screening platform was developed ( FIG.7). The platform used primary human T cells to rapidly identify V HH domains that function as chimeric antigen receptor (CAR) binding motifs. Primary human CD4+and CD8+T cells were activated with anti-CD3 / CD28-coated beads for 48 hours. At 24 hours post-activation, T cells were transduced at a multiplicity of infection (MOI) <1 with lentiviral libraries encoding the desired V HH repertoires derived from post-vaccination samples. After 48 hours, beads and residual virus were removed. Cells were then expanded for five days, after which CAR+T cells were enriched to >95% purity by fluorescence-activated cell sorting ( FACS). Sorted cells were allowed to recover and expand for an additional four days before initiation of screening.

[0293] At the onset of the screen, a day 0 (DO) a sample ( “input sample”) was collected from each screening condition. The CAR T cell libraries enriched as described above were cocultured with target K562 cells constitutively expressing blue fluorescent protein (BF P) and high levels of either CLDN18.1 or CLDN18.2. Effector-to-target (E: T) ratios were maintained at 5:1. Additional conditions included untransduced (UTD) K562 controls and a CAR T-only condition supplemented with IL-7 and IL-15. T cell activation was monitored by measuring CD25 expression and changes in population dynamics. On DO, a subset of cells from each condition was collected for flow cytometry to quantify cell numbers and establish baseline CD25 expression. After three days (D3), a second subset of cells was analyzed to assess CD25 upregulation in functional CAR T cells and to quantify target cell depletion. When target depletion was observed, fresh K562 cells were added to restore the 5: 1 E: T ratio, ensuring continued antigen stimulation. On day 5 (D5), a final subset of cells was collected for flow cytometry, and all remaining cells were harvested for two-way FACS. Residual target cells were excluded using a BF P dump gate, and CAR+T cells (GF P+) were sorted into CD25 and CD25+fractions. In conditions lacking CD25+cells (e.g., CAR T-only or UTD controls), only CD25 cells were collected.

[0294] Genomic DNA (gDNA) was extracted from each sorted population. VHH sequences were amplified by PCR and prepared for next-generation sequencing using an ILLUMINA™ MISEQ™ 600-cycle v3 next-generation sequencing kit (paired-end). To preserve the identity of each library during sequencing, uniquely indexed i7 reverse primers were combined with staggered i5 forward primers to enhance sequence diversity. Sequencing data were processed to identify candidate (“hit”) sequences for subsequent individual validation (e.g., FIG. 10). Ca lcul ation of “hit” scores

[0295] VHH domains considered as candidates for further validation (i.e., “hits”) were broadly determined computationally by calculating the fold change between the CD25+ CAR T cells co-cultured with CLDN18.2 positive target cells divided by the CD25- CAR T cells co-cultured with CLDN18.2 positive target cells. Cells that enrich in the CD25+ condition were considered hits. Given previous data showing that sequences enriched in the vaccination repertoire libraries were more likely to be functionally binders, the starting abundance of a given sequence was strongly weighted.

[0296] A refined “hit score” was calculated in an attempt to incorporate more of the screening data generated and orthogonally validate the fold change by abundance. The hit score was calculated as follows:

[0297] Enrichment score (higher values were favored, indicative of functional binders) = ( (CD25+ / UTD) + (CD25+ / CD25-))

[0298] Noise (higher values were not favored, indicative of noise in the screen or off-target behavior) = ( (Input / UTD) + (T cell only / Input)) Starting Abundance (higher values are valued, indicative of functional binders) = Input

[0299] Hit Score = (Enrichment score - Noise + Starting Abundance)

[0300] The hit score could then be used to calculate statistical significance of a given sequence and its performance in the screen described above.

[0301] Sequences

[0302] What follows is the nucleotide sequence of the chimeric antigen receptor (CAR) plasmid (pSLCAR-VHH) used for cloning of VHH domains, where the nucleotide sequence encoding a chimeric antigen receptor polypeptide linked to enhanced green fluorescent protein (eGF P) polypeptide by way of a self-cleaving peptide ( P2A), and prior to insertion of an anti-CLDN18 VHH domain sequence, is shown in BOLD UPPERCASE TEXT. In the below sequence, sites recognized by the Bpil restriction enzyme are shown as BOLD, UPPERCASE, UN DERL I NED TEXT A map of the plasmid is provided at FIGs. 5A and 5B, and Tables 5A and 5B provide amino acid and polynucleotide sequences corresponding to the features labeled in the plasmid map. gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagc c ca tatatggagttc cgcgttacataacttacggtaaatggc c cgc ctggctgac cgc c caacga c c c c cgc c cattgacgtcaataatgacgtatgttc c catagtaacgc caatagggactttc c attgacgtcaatgggtggagtatttacggtaaactgc c cacttggcagtacatcaagtgtat catatgc caagtacgc c c c ctattgacgtcaatgacggtaaatggc c cgc ctggcattatgc c cagtacatgac cttatgggactttc ctacttggcagtacatctacgtattagtcatcgcta ttac catggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgg ggatttc caagtctc cac c c cattgacgtcaatgggagtttgttttggcac caaaatcaacg ggactttc caaaatgtcgtaacaactc cgc c c cattgacgcaaatgggcggtaggcgtgtac ggtgggaggtctatataagcagcgcgttttgc ctgtactgggtctctctggttagac cagat ctgagc ctgggagctctctggctaactagggaac c cactgcttaagc ctcaataaagcttgc cttgagtgcttcaagtagtgtgtgc c cgtctgttgtgtgactctggtaactagagatc c ctc agac c cttttagtcagtgtggaaaatctctagcagtggcgc c cgaacagggacttgaaagcg aaagggaaac cagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaag aggcgaggggcggcgactggtgagtacgc caaaaattttgactagcggaggctagaaggaga gagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaatt cggttaaggc cagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcaggga gctagaacgattcgcagttaatc ctggc ctgttagaaacatcagaaggctgtagacaaatac tgggacagctacaac catc c cttcagacaggatcagaagaacttagatcattatataataca gtagcaac c ctctattgtgtgcatcaaaggatagagataaaagacac caaggaagctttaga caagatagaggaagagcaaaacaaaagtaagac cac cgcacagcaagcggc cgctgatcttc agac ctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagt aaaaattgaac cattaggagtagcac c cac caaggcaaagagaagagtggtgcagagagaaa aaagagcagtgggaataggagctttgttc cttgggttcttgggagcagcaggaagcactatg ggcgcagcgtcaatgacgctgacggtacaggc cagacaattattgtctggtatagtgcagca gcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggg gcatcaagcagctc caggcaagaatc ctggctgtggaaagatac ctaaaggatcaacagctc ctggggatttggggttgctctggaaaactcatttgcac cactgctgtgc cttggaatgctag ttggagtaataaatctctggaacagatttggaatcacacgac ctggatggagtgggacagag aaattaacaattacacaagcttaatacactc cttaattgaagaatcgcaaaac cagcaagaa aagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacat aacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaa gaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcac cattatcg tttcagac c cac ctc c caac c c cgaggggac c c cgggtttattacagggacagcagagatc c actttggcgc cggctcgagttttaaaagaaaaggggggattggggggtacagtgcaggggaa agaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaa aattcaaaatttttcgagtggctc cggtgc c cgtcagtgggcagagcgcacatcgc c cacag tc c c cgagaagttggggggaggggtcggcaattgaac cggtgc ctagagaaggtggcgcggg gtaaactgggaaagtgatgtcgtgtactggctc cgc ctttttc c cgagggtgggggagaac c gtatataagtgcagtagtcgc cgtgaacgttctttttcgcaacgggtttgc cgc cagaacac aggtgtcgtgacgcgggatc cgc cac cATGCTCAGGCTGCTCT TGGCTCTCAACT TAT TCCC T TCAAT TCAAG TAACAG GAG G G TCT TCCT T T T T T GAAGACCCGACGCCAGCGCCAACTAT T G CGAGTCAGCCTCTCAGTCTGCGACCTGAGGCT TGTCGACCAGCAGCCGGAGGCGCAGTGCAC ACGAGGGGGCTGGACT TCGCCTGTGATCCT TCTAAGCCCT T T TGGGTGCTGGTGGTGGT TGG TGGAGTCCTGGCT TGCTATAGCT TGCTAGTAACAGTGGCCT T TAT TAT T T TCTGGGTGAGGA AACGGGGCAGAAAGAAACTCCTGTATATAT TCAAACAACCAT T TATGAGACCAGTACAAACT ACTCAAGAGGAAGATGGCTGTAGCTGCCGAT T TCCAGAAGAAGAAGAAGGAGGATGTGAACT GGCTAGCCTGAGAGTGAAGT TCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGA ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT T T TGGACAAGAGA CGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGAT TGGGATGAAAGGCG AGCGCCGGAGGGGCAAGGGGCACGATGGCCT T TACCAGGGTCTCAGTACAGCCACCAAGGAC ACCTACGACGCCCT TCACATGCAGGCCCTGCCCCCTCGCGCTAGCGCCACGAACT TCTCTCT GT TAAAGCAAGCAGGCGACGTGGAAGAAAACCCCGGTCCCGTGAGCAAGGGCGAGGAGCTGT TCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT TCAGC GTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGT TCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGT GCT TCAGCCGCTACCCCGACCACATGAAGCAGCACGACT TCT TCAAGTCCGCCATGCCCGAA GGCTACGTCCAGGAGCGCACCATCT TCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGA GGTGAAGT TCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACT TCAAGG AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACT TCAAGATCCGCCACAACATCGAGGA CGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGC TGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAG CGCGATCACATGGTCCTGCTGGAGT TCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA GCTGTACAAGtaacgcgttaagtcgacaatcaac ctctggattacaaaatttgtgaaagatt gactggtattcttaactatgttgctc cttttacgctatgtggatacgctgctttaatgc ctt tgtatcatgctattgcttc c cgtatggctttcattttctc ctc cttgtataaatc ctggttg ctgtctctttatgaggagttgtggc c cgttgtcaggcaacgtggcgtggtgtgcactgtgtt tgctgacgcaac c c c cactggttggggcattgc cac cac ctgtcagctc ctttc cgggactt tcgctttc c c c ctc c ctattgc cacggcggaactcatcgc cgc ctgc cttgc c cgctgctgg acaggggctcggctgttgggcactgacaattc cgtggtgttgtcggggaaatcatcgtc ctt tc cttggctgctcgc ctgtgttgc cac ctggattctgcgcgggacgtc cttctgctacgtc c cttcggc c ctcaatc cagcggac cttc cttc c cgcggc ctgctgc cggctctgcggc ctctt c cgcgtctttgc cttcgc c ctcagacgagtcggatctc c ctttgggc cgc ctc c c cgcgtcg actttaagac caatgacttacaaggcagctgtagatcttagc cactttttaaaagaaaaggg gggactggaagggctaattcactc c caacgaagataagatctgctttttgcttgtactgggt ctctctggttagac cagatctgagc ctgggagctctctggctaactagggaac c cactgctt aagc ctcaataaagcttgc cttgagtgcttcaagtagtgtgtgc c cgtctgttgtgtgactc tggtaactagagatc c ctcagac c cttttagtcagtgtggaaaatctctagcagtacgtata gtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagt gagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaattt cacaaataaagcatttttttcactgcattctagttgtggtttgtc caaactcatcaatgtat cttatcatgtctggctctagctatc c cgc c c ctaactc cgc c catc c cgc c c ctaactc cgc c cagttc cgc c cattctc cgc c c catggctgactaattttttttatttatgcagaggc cgag gc cgc ctcggc ctctgagctattc cagaagtagtgaggaggcttttttggaggc ctagggac gtac c caattcgc c ctatagtgagtcgtattacgcgcgctcactggc cgtcgttttacaacg tcgtgactgggaaaac c ctggcgttac c caacttaatcgc cttgcagcacatc c c c ctttcg c cagctggcgtaatagcgaagaggc c cgcac cgatcgc c cttc c caacagttgcgcagc ctg aatggcgaatgggacgcgc c ctgtagcggcgcattaagcgcggcgggtgtggtggttacgcg cagcgtgac cgctacacttgc cagcgc c ctagcgc c cgctc ctttcgctttcttc c cttc ct ttctcgc cacgttcgc cggctttc c c cgtcaagctctaaatcgggggctc c ctttagggttc cgatttagtgctttacggcac ctcgac c c caaaaaacttgattagggtgatggttcacgtag tgggc catcgc c ctgatagacggtttttcgc c ctttgacgttggagtc cacgttctttaata gtggactcttgttc caaactggaacaacactcaac c ctatctcggtctattcttttgattta taagggattttgc cgatttcggc ctattggttaaaaaatgagctgatttaacaaaaatttaa cgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcg cggaac c c ctatttgtttatttttctaaatacattcaaatatgtatc cgctcatgagacaat aac c ctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttc cgt gtcgc c cttattc c cttttttgcggcattttgc cttc ctgtttttgctcac c cagaaacgct ggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatc tcaacagcggtaagatc cttgagagttttcgc c c cgaagaacgttttc caatgatgagcact tttaaagttctgctatgtggcgcggtattatc c cgtattgacgc cgggcaagagcaactcgg tcgc cgcatacactattctcagaatgacttggttgagtactcac cagtcacagaaaagcatc ttacggatggcatgacagtaagagaattatgcagtgctgc cataac catgagtgataacact gcggc caacttacttctgacaacgatcggaggac cgaaggagctaac cgcttttttgcacaa catgggggatcatgtaactcgc cttgatcgttgggaac cggagctgaatgaagc catac caa acgacgagcgtgacac cacgatgc ctgtagcaatggcaacaacgttgcgcaaactattaact ggcgaactacttactctagcttc c cggcaacaattaatagactggatggaggcggataaagt tgcaggac cacttctgcgctcggc c cttc cggctggctggtttattgctgataaatctggag c cggtgagcgtgggtctcgcggtatcattgcagcactggggc cagatggtaagc c ctc c cgt atcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgc tgagataggtgc ctcactgattaagcattggtaactgtcagac caagtttactcatatatac tttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc ctttttgat aatctcatgac caaaatc c cttaacgtgagttttcgttc cactgagcgtcagac c c cgtaga aaagatcaaaggatcttcttgagatc ctttttttctgcgcgtaatctgctgcttgcaaacaa aaaaac cac cgctac cagcggtggtttgtttgc cggatcaagagctac caactctttttc cg aaggtaactggcttcagcagagcgcagatac caaatactgttcttctagtgtagc cgtagtt aggc cac cacttcaagaactctgtagcac cgc ctacatac ctcgctctgctaatc ctgttac cagtggctgctgc cagtggcgataagtcgtgtcttac cgggttggactcaagacgatagtta c cggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagc c cagcttggagcg aacgac ctacac cgaactgagatac ctacagcgtgagctatgagaaagcgc cacgcttc c cg aagggagaaaggcggacaggtatc cggtaagcggcagggtcggaacaggagagcgcacgagg gagcttc cagggggaaacgc ctggtatctttatagtc ctgtcgggtttcgc cac ctctgact tgagcgtcgatttttgtgatgctcgtcaggggggcggagc ctatggaaaaacgc cagcaacg cggc ctttttacggttc ctggc cttttgctggc cttttgctcacatgtgtcgacggatcggg agatctc c cgatc c c ctatggtgcactctcagtacaatctgctctgatgc cgcatagttaag c cagtatctgctc c ctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaag ctacaacaaggcaaggcttgac cgacaattgcatgaagaatctgcttagggttaggcgtttt gcgctgcttcgcgatgtacgggc cagatatacgcgtt

[0303] What follows is the amino acid sequence encoded by the BOLD ALL CAPS sequence shown above, which corresponds to a CAR sequence lacking an antigen binding domain (e.g., a VHH domain) that is linked to enhanced green fluorescent protein (eGF P) polypeptide by way of a self-cleaving peptide (P2A).

[0304] M LRL L LAL N L F P S IQVTGGS S F FEDPTPAPTIASQP L S LRPEACRPAAGGAV HTRGLDFACD P SKP F W V L V WGGV LACYS L L VTVAF I I F W VRKRGRKKL LYI FKQP F MRP VQTTQEEDGCSC RF PEEEEGGCELAS LRVKF SRSADAPAYQQGQNQLYNEL N LGRREEYDV LDKRRGRDPEMGG KPQRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDAL H MQA L P PRASATN F S L LKQAGDVEEN PGP V SKGEEL FTGW P I L VELDGDV NGHKF S V SGEGEGDA TYGKLTLKF ICTTGKL P V P W PTL VTTLTYGVQCF SRYPDH MKQHDF FKSAM PEGYVQERTI F FKDDGNYKTRAEVKFEGDTL V NRIELKGIDFKEDGN I LGHKLEYNYN S H N VYI MADKQKNGI KV N FKIRH N IEDGS VQLADHYQQNTP IGDGP V L L PDN HYL STQSAL SKDP NEKRDH M V L LEF VTAAGITLGMDELYK

[0305] What follows is the nucleotide sequence of the plasmid encoding the activation reporter cassette used in the Examples. A map of the plasmid is provided at FIGs. 6A and 6B, and Tables 5A and 5B provide amino acid and polynucleotide sequences corresponding to the features labeled in the plasmid map. ggc cgc cagcacagtggtcgatcgacgataaaataaaagattttatttagtctc cagaaaaa ggggggaatgaaagac c c cac ctgtaggtttggcaagctagcttaagtaacgc cattttgca aggcatggaaaaatacataactgagaatagaaaagttcagatcaaggtcaggaacagatgga acagggtcgcgtc c cgcaataaaagagc c cacaac c c ctcactcggggcgc cagtc ctc cga ttgactgagtcgc c cgggtac c cgtgtatc caataaac c ctcttgcagttgcatc cgacttg tggtctcgctgttc cttgggagggtctc ctctgagtgattgactac c cgtcagcgggggtct ttcacatgcagcatgtatcaaaattaatttggttttttttcttaagtatttacattaaatgg c catagtagttcattatggacagcgcagaaagagctggggagaattgtgaaattgttatc cg ctcacaattc cacacaacatacgagc cggaagcataaagtgtaaagc ctggggtgc ctaatg agtgagctaactcacattaattgcgttgcgctcactgc c cgctttc cagtcgggaaac ctgt cgtgc cagctgcattaatgaatcggc caacgcgcggggagaggcggtttgcgtattgggcgc tcttc cgcttc ctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatc agctcactcaaaggcggtaatacggttatc cacagaatcaggggataacgcaggaaagaaca tgtgagcaaaaggc cagcaaaaggc caggaac cgtaaaaaggc cgcgttgctggcgtttttc cataggctc cgc c c c c ctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaa c c cgacaggactataaagatac caggcgtttc c c c ctggaagctc c ctcgtgcgctctc ctg ttc cgac c ctgc cgcttac cggatac ctgtc cgc ctttctc c cttcgggaagcgtggcgctt tctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctc caagctgggctg tgtgcacgaac c c c c cgttcagc c cgac cgctgcgc cttatc cggtaactatcgtcttgagt c caac c cggtaagacacgacttatcgc cactggcagcagc cactggtaacaggattagcaga gcgaggtatgtaggcggtgctacagagttcttgaagtggtggc ctaactacggctacactag aagaacagtatttggtatctgcgctctgctgaagc cagttac cttcggaaaaagagttggta gctcttgatc cggcaaacaaac cac cgctggtagcggtggtttttttgtttgcaagcagcag attacgcgcagaaaaaaaggatctcaagaagatc ctttgatcttttctacggggtctgacgc tcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttca c ctagatc cttttgcggc cggc cgcaaatcaatctaaagtatatatgagtaaacttggtctg acagttac caatgcttaatcagtgaggcac ctatctcagcgatctgtctatttcgttcatc c atagttgc ctgactc c c cgtcgtgtagataactacgatacgggagggcttac catctggc c c cagtgctgcaatgatac cgcgagac c cacgctcac cggctc cagatttatcagcaataaac c agc cagc cggaagggc cgagcgcagaagtggtc ctgcaactttatc cgc ctc catc cagtct attaattgttgc cgggaagctagagtaagtagttcgc cagttaatagtttgcgcaacgttgt tgc cattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctc cg gttc c caacgatcaaggcgagttacatgatc c c c catgttgtgcaaaaaagcggttagctc c ttcggtc ctc cgatcgttgtcagaagtaagttggc cgcagtgttatcactcatggttatggc agcactgcataattctcttactgtcatgc catc cgtaagatgcttttctgtgactggtgagt actcaac caagtcattctgagaatagtgtatgcggcgac cgagttgctcttgc c cggcgtca atacgggataatac cgcgc cacatagcagaactttaaaagtgctcatcattggaaaacgttc ttcggggcgaaaactctcaaggatcttac cgctgttgagatc cagttcgatgtaac c cactc gtgcac c caactgatcttcagcatcttttactttcac cagcgtttctgggtgagcaaaaaca ggaaggcaaaatgc cgcaaaaaagggaataagggcgacacggaaatgttgaatactcatact cttc ctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatat ttgaatgtatttagaaaaataaacaaataggggttc cgcgcacatttc c c cgaaaagtgc ca c cagctttgctcttaggagtttc ctaatacatc c caaactcaaatatataaagcatttgact tgttctatgc c ctagttattaatagtaatcaattacggggtcattagttcatagc c catata tggagttc cgcgttacataacttacggtaaatggc c cgc ctggctgac cgc c caacgac c c c cgc c cattgacgtcaataatgacgtatgttc c catagtaacgc caatagggactttc cattg acgtcaatgggtggagtatttacggtaaactgc c cacttggcagtacatcaagtgtatcata tgc caagtacgc c c c ctattgacgtcaatgacggtaaatggc c cgc ctggcattatgc c cag tacatgac cttatgggactttc ctacttggcagtacatctacgtattagtcatcgctattac catggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggat ttc caagtctc cac c c cattgacgtcaatgggagtttgttttggcac caaaatcaacgggac tttc caaaatgtcgtaacaactc cgc c c cattgacgcaaatgggcggtaggcgtgtacggtg ggaggtctatataagcagagctcaataaaagagc c cacaac c c ctcactcggcgcgc cagtc ctc cgattgactgagtcgc c cgggtac c cgtgtatc caataaac c ctcttgcagttgcatc c gacttgtggtctcgctgttc cttgggagggtctc ctctgagtgattgactac c cgtcagcgg gggtctttcatttgggggctcgtc cgggatcgggagac c c ctgc c cagggac cac cgac c ca c cac cgggaggtaagctggc cagcaacttatctgtgtctgtc cgattgtctagtgtctatga ctgattttatgcgc ctgcgtcggtactagttagctaactagctctgtatctggcggac c cgt ggtggaactgacgagttcggaacac c cggc cgcaac c ctgggagacgtc c cagggacttcgg gggc cgtttttgtggc c cgac ctgagtc caaaaatc c cgatcgttttggactctttggtgca c c c c c cttagaggagggatatgtggttctggtaggagacgagaac ctaaaacagttc c cgc c tc cgtctgaatttttgctttcggtttgggac cgaagc cgcgc cgcgcgtcttgtctgctgca gcatcgttctgtgttgtctctgtctgactgtgtttctgtatttgtctgaaaatatgggc c cg ggc cagactgttac cactc c cttaagtttgac cttaggtcactggaaagatgtcgagcggat cgctcacaac cagtcggtagatgtcaagaagagacgttgggttac cttctgctctgcagaat ggc caac ctttaacgtcggatggc cgcgagacggcac ctttaac cgagac ctcatcac c cag gttaagatcaaggtcttttcac ctggc c cgcatggacac c cagac caggtc c c ctacatcgt gac ctgggaagc cttggcttttgac c c c c ctc c ctgggtcaagc c ctttgtacac c ctaagc ctc cgc ctc ctcttc ctc catc cgc c c cgtctctc c c c cttgaac ctc ctcgttcgac c c cg c ctcgatc ctc c ctttatc cagc c ctcactc cttctctaggcgc c c c catatggc catatga gatcttatatggggcac c c c cgc c c cttgtaaacttc c ctgac c ctgacatgacaagagtta ctaacagc c c ctctctc caagctcacttacaggctctctacttagtc cagcacgaagtctgg agac ctctggcggcagc ctac caagaacaactggac cgac cggtggtac ctcac c cttac cg agtcggcgacacagtgtgggtc cgc cgacac cagactaagaac ctagaac ctcgctggaaag gac cttacacagtc ctgctgac cac c c c cac cgc c ctcaaagtagacggcatcgcagcttgg atacacgc cgc c cacgtgaaggctgc cgac c c cgggggtggac catc ctctagactgc cgga tc caagctggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacaga aggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaag gcgtcgcgaattcgcggagactctagagggtatataatggaagctcgatttc cagcttggca ttc cggtactgttggtaaacac caagctatgac cgagtacaagc c cacggtgcgc ctcgc ca c c cgcgacgacgtc c c cagggc cgtacgcac c ctcgc cgc cgcgttcgc cgactac c c cgc c acgcgc cacac cgtcgatc cggac cgc cacatcgagcgggtcac cgagctgcaagaactctt c ctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgc cgc cgtgg cggtctggac cacgc cggagagcgtcgaagcgggggcggtgttcgc cgagatcggc c cgcgc a tggc cgagtt gagegg ttc c cggctggc cgcgcagcaacagatggaaggc ctc ctggcgc c gcac cggc c caaggagc c cgcgtggttc ctggc cac cgtcggcgtttcgc c cgac cac cagg gcaagggtctgggcagcgc cgtcgtgctc c c cggagtggaggcggc cgagcgcgc cggggtg c c cgc cttc ctggagac ctc cgcgc c c cgcaac ctc c c cttctacgagcggctcggcttcac cgtcac cgc cgacgtcgaggtgc c cgaaggac cgcgcac ctggtgcatgac c cgcaagc c cg gtgc cgctagc cgggc caagcggtc cggatc cggacagtgcac caactatgcgctgctgaaa ctggcgggcgatgtggaaagcaac c cgggc c cgatggcttcgtac c c ctgc catcaacacgc gtctgcgttcgac caggctgcgcgttctcgcggc catagcaac cgacgtacggcgttgcgc c ctcgc cggcagcaagaagc cacggaagtc cgc ctggagcagaaaatgc c cacgctactgcgg gtttatatagacggtc ctcacgggatggggaaaac cac cac cacgcaactgctggtggc c ct gggttcgcgcgacgatatcgtctacgtac c cgagc cgatgacttactggcaggtgctggggg cttc cgagacaatcgcgaacatctacac cacacaacac cgc ctcgac cagggtgagatatcg gc cggggacgcggcggtggtaatgacaagcgc c cagataacaatgggcatgc cttatgc cgt gac cgacgc cgttctggctc ctcatatcgggggggaggctgggagctcacatgc c c cgc c c c cggc c ctcac c ctcatcttcgac cgc catc c catcgc cgc c ctc ctgtgctac c cggc cgcg cgatac cttatgggcagcatgac c c c c caggc cgtgctggcgttcgtggc c ctcatc c cgc c gac cttgc c cggcacaaacatcgtgttgggggc c cttc cggaggacagacacatcgac cgc c tggc caaacgc cagcgc c c cggcgagcggcttgac ctggctatgctggc cgcgattcgc cgc gtttacgggctgcttgc caatacggtgcggtatctgcagggcggcgggtcgtggcgggagga ttggggacagctttcggggacggc cgtgc cgc c c cagggtgc cgagc c c cagagcaacgcgg gc c cacgac c c catatcggggacacgttatttac c ctgtttcgggc c c c cgagttgctggc c c c caacggcgac ctgtacaacgtgtttgc ctgggc cttggacgtcttggc caaacgc ctc cg tc c catgcacgtctttatc ctggattacgac caatcgc c cgc cggctgc cgggacgc c ctgc tgcaacttac ctc cgggatggtc cagac c cacgtcac cac c c c cggctc catac cgacgatc tgcgac ctggcgcgcacgtttgc c cgggagatgggggaggctaac cgcgc caagcgctcggg ttcgggtgc cac caacttcagc ctgctgaagcaggc cggcgacgtggaggagaac c c cggc c c catggtgagcgagctgattaaggagaacatgcacatgaagctgtacatggagggcac cgtg aacaac cac cacttcaagtgcacatc cgagggcgaaggcaagc c ctacgagggcac c cagac catgagaatcaaggcggtcgagggcggc c ctctc c c cttcgc cttcgacatc ctggctac ca gcttcatgtacggcagcaaaac cttcatcaac cacac c cagggcatc c c cgacttctttaag cagtc cttc c c cgagggcttcacatgggagagagtcac cacatacgaagacgggggcgtgct gac cgctac c caggacac cagc ctc caggacggctgc ctcatctacaacgtcaagatcagag gggtgaacttc c catc caacggc c ctgtgatgcagaagaaaacactcggctgggaggc ctc c ac cgagac c ctgtac c c cgctgacggcggc ctggaaggcagagc cgacatggc c ctgaagct cgtgggcgggggc cac ctgatctgcaacttgaagac cacatacagatc caagaaac c cgcta agaac ctcaagatgc c cggcgtctactatgtggacagaagactggaaagaatcaaggaggc c gacaaagagac ctacgtcgagcagcacgaggtggctgtggc cagatactgcgac ctc c ctag caaactggggcacagataa

[0306] Table 5A. Nucleotide sequences for plasmid features.

[0307]

[0308]

[0309]

[0310]

[0311]

[0312] Table 5B. Amino acid sequences for polypeptides encoded by plasmid features.

[0313]

[0314]

[0315] Table 6A. Sequences fo r cluster 0 a n ti-CLD N18.2 VH H a n tibodies.

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326] Table 6B. Sequences fo r cluster 1 a n ti-CLD N18.2VH H a n tibodies.

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338] Table 6C. Sequences for cluster 8 anti-CLDN18.2 VHH antibodies

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349] Table 6D. Sequences for cluster 10 anti-CLDN18.2 VHH antibodies.

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] Table 6E. Sequences for cluster 11 anti-CLDN18.2 VHH antibodies.

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387] Table 6F. Sequences for cluster 12 anti-CLD N18.2VHH antibodies.

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398] Table 6G. Sequences fo r cluster 13 a n ti-CLD N18.2 VH H a n tibodies.

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417] Table 6H. Sequences for cluster 14 anti-CLDN18.2 VHH antibodies.

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432] Table 61. Sequences for cluster 15 anti-CLDN18.2 VHH antibodies.

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454] Table 6K. Sequences for cluster 17 anti-CLD18.2 VHH antibodies.

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468] Table 6L. Sequences for clusters 18 and 19 anti-CLDN18.2 VHH antibodies.

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483] Table 6M. Sequences for clusters 2 to 5 of anti-CLDN18.2 VHH antibodies.

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549] Table 6N. Sequences fo r clusters 6, 7, a nd 9 of a n tiCLD N18.2 VH H a n tibodies.

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579] Table 7. Consensus sequences for complementa rity determining regions of anti-CLDN18.2 VHH antibody sequences fal ling within each cluster of Tables 6A to 6N.

[0580]

[0581] Table 8. Amino acid sequences for representative anti-CLDN18 VHH antibodies.

[0582]

[0583]

[0584] Other Embodiments

[0585] From the foregoing description, it will be apparent that variations and modifications may be made to the various aspects and embodiments described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0586] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0587] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A VH-heavy chain only (VHH) polypeptide or an antigen binding portion thereof that specifically binds to a Claudin-18.1 (CLDN18.1) and / or Claudin-18.2 (CLDN18.2) polypeptide or a fragment thereof, wherein the VHH polypeptide or the antigen binding portion thereof comprises three Complementarity Determining Regions (CDRs): CDR1, CDR2 and CDR3, which are structurally positioned between four camelid VHH framework (FR) regions (FR1-FR4) as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; wherein the three CDRs are selected from:CDR1 comprising the amino acid sequence GRTFSSYA; CDR2 comprising the amino acid sequence ISWSGGKT; and CDR3 comprising an amino acid sequence selected from the group consisting of AAERDLLWLRYWDY; VAERDLLWLRYWDY; AAERDLRWLRYWDY; AAERDLLWLRYVVDD; AAERDLLLLRYWDY; AAERDLLWLRDVVDY;AAERDRLWLRYWDY; AAERNLLWLRYVVDY; ATERDLLWLRYWDY; and AGERDLLWLRYWDY;CDR1 comprising the amino acid sequence GRTFSSYI; CDR2 comprising the amino acid sequence ISWSGDST; and CDR3 comprising the amino acid sequence AAGRTTWANYAMDY;CDR1 comprising the amino acid sequence GRTFSSYI; CDR2 comprising the amino acid sequence ISWSGGST; and CDR3 comprising the amino acid sequence AAGSTTWATYSMDY;CDR1 comprising the amino acid sequence GRTLSSFA; CDR2 comprising the amino acid sequence ISWSGGIT; and CDR3 comprising the amino acid sequence AAGSTTWATYSMDY.

2. The VHH polypeptide of claim 1, wherein the VHH polypeptide or antigen binding portion thereof selectively binds to CLDN18.2.

3. The VHH polypeptide of claim 1, whereini) FR1 comprises the following amino acid sequence:QQLVESGGGLXiQAGGSLRLSCAAS, whereinXi is A or V;ii) FR2 comprises the following amino acid sequence:MGWFRQAPGKEREXaVAXb, whereinXais F or V; andXb is A or G;iii) FR3 comprises the following amino acid sequence:YYXcDSXdKGRFTISRDNXeKNTVYLQMNSLKXfEDTAVYYC, whereinXcis A or D;Xais V or L;Xeis A or G; andXf is T or P; andiv) FR4 comprises the following amino acid sequence: WGQGTSVTVSSPTPAPTI.

4. A polypeptide that specifically binds to Claudin-18.1 (CLDN18.1) or Claudin-18.2 (CLDN18.2), wherein the polypeptide or a CLDN18.1 or CLDN-18.2 binding portion thereof has at least 85% amino acid sequence identity to a sequence selected from the group consisting of:VQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGKTYYADS LKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAERDLLWLRYVVDYWGQGTSVTVSSPTP APTI;VQLVESGGGLVQAGGSLRLSCAASGRTFSSYIMGWFRQAPGKEREVVAGISWSGDSTYYADS VKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCAAGRTTVVANYAMDYWGQGTSVTVSSPTP APTI;VQLVESGGGLVQAGGSLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAISWSGGSTYYDDS VKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGSTTVVATYSMDYWGQGTSVTVSSPTP APTI; and VQLVESGGGLAQAGGSLRLSCAASGRTLSSFAMGWFRQAPGKEREFVAAISWSGGITYYADS VKGRFTISRDNAKNTVYLQMNSLKTEDTAVYYCAAGSTTWATYSMDYWGQGTSVTVSSPTP APTI.

5. A chimeric antigen receptor polypeptide (CAR) comprising the polypeptide of any one of claims 1-4.

6. The CAR of claim 5, wherein the CAR comprises in order from N-terminus to C-terminus a CD28 signal peptide, the polypeptide of any one of claims 1-4, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 cytoplasmic domain, and a CD3(^ domain.

7. A polynucleotide encoding the VHH polypeptide of any one of claims 1-4.

8. A polynucleotide encoding the CAR of claim 5 or claim 6.

9. A vector comprising the polynucleotide of claim 7 or claim 8.

10. The vector of claim 9, wherein the vector is an expression vector.

11. A cell comprising the polynucleotide of claim 7 or claim 8 or the vector of claim 9 or claim 10.

12. The cell of claim 11, wherein the cell is an immune cell.

13. The cell of claim 12, wherein the immune cell is a T cell.

14. The cell of any one of claims 11-13, wherein the cell is in vitro, ex vivo, or in vivo.

15. A pharmaceutical composition comprising an effective amount of the polypeptide of any one of claims 1-4, or a CLDN18.1 or CLDN18.2 binding fragment thereof, the CAR of claim 5 or claim 6, the polynucleotide of claim 7 or claim 8, the vector of claim 9 or claim 10, or the cell of any one of claims 11-14, and a pharmaceutically acceptable excipient, carrier, or diluent.

16. A method for treating a subject having a neoplasia, the method comprising administering to the subject the pharmaceutical composition of claim 15.

17. A kit comprising the polypeptide of any one of claims 1-4, or a CLDN18.1 or CLDN18.2 binding fragment thereof, the CAR of claim 5 or claim 6, the polynucleotide of claim 7 or claim 8, the vector of claim 9 or claim 10, the cell of any one of claims 11-14, orthe pharmaceutical composition of claim 15, and a container, for use in treating a subject having a neoplasia.

18. The method of claim 16 or claim 17, wherein the neoplasia is a stomach cancer, a pancreatic cancer, an esophageal cancer, or an ovarian cancer.

19. A method of detecting Claudin-18.1 (CLDN18.1) and / or Claudin-18.2 (CLDN18.2) polypeptide or a fragment thereof in a sample, the method comprising:contacting the sample with at least one detectably labeled VH-heavy chain only (VHH) polypeptide or an antigen binding fragment thereof that specifically binds to CLDN18.1 and / or CLDN18.2 or a or a fragment thereof, wherein the VHH polypeptide or the antigen binding fragment thereof comprises three Complementarity Determining Regions (CDRs), CDR1, CDR2, and CDR3 structurally positioned between four framework (FR) regions (FR1-FR4) as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; wherein the three CDRs are selected from:CDR1 comprising the amino acid sequence GRTFSSYA; CDR2 comprising the amino acid sequence ISWSGGKT; and CDR3 comprising an amino acid sequence selected from the group consisting of AAERDLLWLRYWDY; VAERDLLWLRYWDY; AAERDLRWLRYWDY; AAERDLLWLRYVVDD; AAERDLLLJLJRYWDY; AAERDLLWLRDWDY;AAERDRLWLRYWDY; AAERNLLWLRYVVDY; ATERDLLWLRYWDY; and AGERDLLWLRYWDY;CDR1 comprising the amino acid sequence GRTFSSYI; CDR2 comprising the amino acid sequence ISWSGDST; and CDR3 comprising the amino acid sequence AAGRTTWANYAMDY;CDR1 comprising the amino acid sequence GRTFSSYI; CDR2 comprising the amino acid sequence ISWSGGST; and CDR3 comprising the amino acid sequence AAGSTTWATYSMDY;CDR1 comprising the amino acid sequence GRTLSSFA; CDR2 comprising the amino acid sequence ISWSGGIT; and CDR3 comprising the amino acid sequence AAGSTTWATYSMDY; and whereini) FR1 comprises the following amino acid sequence:QQLVESGGGLXiQAGGSLRLSCAAS, whereinXi is A or V;ii) FR2 comprises the following amino acid sequence:MGWFRQAPGKEREXaVAXb, whereinXais F or V; andXb is A or G;iii) FR3 comprises the following amino acid sequence:YYXcDSXdKGRFTISRDNXeKNTVYLQMNSLKXfEDTAVYYC, whereinXcis A or D;Xais V or L;Xeis A or G; andXf is T or P; andiv) FR4 comprises the following amino acid sequence: WGQGTSVTVSSPTPAPTI; andmeasuring the level of binding of the binding protein to the CLDN18.1 and / or CLDN18.2 polypeptide in the sample relative to a control to detect or identify the presence of CLDN18.1 and / or CLDN18.2 in the sample.

20. The method of claim 19, wherein the sample is selected from biopsy, blood, peripheral blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, tears, stool, or synovial fluid.

21. The method of claim 19, wherein the CLDN18.1 or CLDN18.2 polypeptide or fragment thereof is on the surface of a cell.

22. The method of claim 21, wherein the CLDN18.1 or CLDN18.2 polypeptide or fragment thereof is endogenously expressed by the cell.

23. A VH-heavy chain only (VHH) polypeptide or an antigen binding portion thereof that specifically binds to CLDN18.1 and / or CLDN18.2, wherein the binding protein or the antigen binding portion thereof comprises an amino acid sequence with at least about 95% identity to one of the following sequences:VQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGKTYYADS LKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAERDLLWLRYVVDYWGQGTSVTVSSPTP APTI;VQLVESGGGLVQAGGSLRLSCAASGRTFSSYIMGWFRQAPGKEREWAGISWSGDSTYYADS VKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCAAGRTTVVANYAMDYWGQGTSVTVSSPTP APTI;VQLVESGGGLVQAGGSLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAISWSGGSTYYDDS VKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGSTTVVATYSMDYWGQGTSVTVSSPTP APTI; and VQLVESGGGLAQAGGSLRLSCAASGRTLSSFAMGWFRQAPGKEREFVAAISWSGGITYYADS VKGRFTISRDNAKNTVYLQMNSLKTEDTAVYYCAAGSTTVVATYSMDYWGQGTSVTVSSPTP APTI.

24. A polynucleotide encoding the VH-heavy chain only (VHH) polypeptide of claim 23.

25. A vector comprising the polynucleotide of claim 24.

26. A VH-heavy chain only (VHH) polypeptide comprising an amino acid sequence having at least 85% sequence identity to a full-length amino acid sequence of any one of Tables 1, 6A to 6N, and 8, or a functional fragment thereof, and / or comprising a set of Complementarity Determining Regions CDR1, CDR2, and CDR3, selected from those listed in any one of Tables 2, 6A to 6N, and 8.

27. The VHH polypeptide of claim 26, wherein the VHH polypeptide, or functional fragment thereof, comprises a set of Complementarity Determining Regions: CDR1, CDR2, and CDR3, listed in a row of any one of Tables 2, 6A to 6N, and 8.

28. A VH-heavy chain only (VHH) polypeptide comprising a full-length amino acid sequence listed in any one of Tables 1, 6 A to 6N, and 8.

29. A polynucleotide encoding the VH-heavy chain only (VHH) polypeptide of any one of claims 26-28.

30. A method for identifying a VHH antibody that binds a target antigen, the method comprising:(a) preparing a library of expression vectors encoding chimeric antigen receptors (CARs), each comprising a VHH domain generated in response to an antigen of interest;(b) expressing each member of the library of expression vectors in a human primary T cell;(c) contacting the human primary T cells with cells that do not express an antigen that is not the antigen of interest and sorting the human primary T cells into CD25+ and CD25-fractions and determining the sequences of VHH domains in each fraction to identify VHH domains enriched in the CD25- fraction; and(d) contacting the immune cell with cells expressing the antigen of interest, sorting the human primary T cells into CD25+ and CD25- fractions, and determining the sequences of VHH domains in each fraction to identify VHH domains enriched in the CD25+ fraction, (e) identifying VHH domain sequences both enriched in the CD25+ fraction of (d) and in the CD25- fraction of (c), thereby identifying VHH domains that selectively bind the target antigen.

31. A method for identifying a VHH antibody that binds a target antigen, the method comprising:(a) preparing a library of expression vectors encoding chimeric antigen receptors (CARs), each comprising a VHH domain generated in response to an antigen of interest;(b) expressing each member of the library of expression vectors in a human primary T cell;(c) contacting the immune cell with cells expressing the antigen of interest, sorting the human primary T cells into CD25+ and CD25- fractions, and determining the sequences of VHH domains in each fraction to identify VHH domains enriched in the CD25+ fraction, thereby identifying VHH domains that bind the target antigen.

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