Claudin-6 binding moiety and its use

Abstract

The present disclosure provides an anti-claudin-6 antibody (e.g., a VHH domain antibody) and a chimeric antigen receptor (CAR) that binds to claudin-6. The chimeric antigen receptor includes an anti-claudin-6 antibody in an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Immune effector cells transduced with the disclosed CAR constructs can be used for cancer immunotherapy.

Description

Technical Field

[0001] Cross-reference This application claims the priority of International Patent Application No. PCT / CN2022 / 109077 filed on July 29, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application incorporates by reference the Sequence Listing submitted together with this application, which is in XML file format, entitled "IEC230078PCT-seql.xml", created on July 26, 2023, and has a size of 68,969 bytes.

[0003] 1. Field The present disclosure relates to the field of antibodies (e.g., domain antibodies), chimeric antigen receptors, and engineered immune cells targeting Claudin-6, and methods of using the same.

Background Art

[0004] 2. Background Claudin (CLDN) is an important tight junction protein that is mainly expressed in a tissue-specific manner in endothelial cells or epithelial cells (see Int J Mol Sci, 14;22(24):13416(2021) (Non-Patent Document 1)). As a member of the CLDN family, claudin-6 (CLDN6) has been found to play an important role in the formation of barriers, particularly the lung epithelial barrier and the epidermal permeability barrier (EPB). Changes in CLDN6 expression are associated with the progression of various cancers, and the malignant phenotypes of tumors affected by CLDN6 include proliferation and apoptosis, migration and invasion, and drug resistance, all of which have been proven to be controlled by important signaling pathways mediated by CLDN6. CLDN6 is a cancer fetal target, which has been proven to be highly expressed in some solid tumors but not in normal tissues (see Histopathology, 61(6):1043-56(2021) (Non-Patent Document 2), Sci Transl Med., 13(579):eabb6282(2021) (Non-Patent Document 3)). The same expression profile has been shown in another cancer fetal target, GPC3. In this technical field, there is a need for new anti-claudin-6 binding agents and immunotherapies, such as CAR-T therapy targeting claudin-6.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0006] 3. Summary In one aspect, the present specification provides an anti-claudin-6 (CLDN6) single-domain antibody (sdAb) that comprises (i) CDR1, CDR2, and CDR3, each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 7, respectively; (ii) CDR1, CDR2, and CDR3, each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 8, respectively; (iii) CDR1, CDR2, and CDR3, each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 9, respectively; (iv) CDR1, CDR2, and CDR3, each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 10, respectively; or (v) CDR1, CDR2, and CDR3, each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 11, respectively. In some aspects, CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof.

[0007] In one aspect, the present specification provides an anti-claudin-6 single-domain antibody (sdAb) that comprises (i) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or (ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 2, CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0008] In some aspects, the anti-claudin-6 sdAb according to the present specification further comprises one or more FR regions shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and / or SEQ ID NO: 11.

[0009] In some embodiments, the present specification provides an anti-claudin-6 sdAb, which comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11. In some embodiments, the present specification provides an anti-claudin-6 sdAb, which comprises, or consists of, an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

[0010] In some embodiments, the anti-claudin-6 sdAb is a camelid sdAb. In some embodiments, the anti-claudin-6 sdAb is a humanized sdAb.

[0011] In some embodiments, the anti-claudin-6 sdAb is genetically fused or chemically conjugated to an agent.

[0012] In another aspect, the present specification provides a fusion protein, which comprises an anti-claudin-6 sdAb according to the present specification and an Fc region (e.g., human IgG1 Fc). In some embodiments, the human IgG1 Fc comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 21-25.

[0013] In another aspect, the present specification provides a chimeric antigen receptor (CAR), which comprises an extracellular antigen-binding domain comprising an anti-claudin-6 sdAb according to the present specification, a transmembrane domain, and an intracellular signaling domain.

[0014] In some embodiments, the extracellular antigen-binding domain of the chimeric antigen receptor further comprises one or more additional antigen-binding domains. In some embodiments, at least one of these additional binding domains binds to GPC3. In some embodiments, the additional antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence of HCDR1, HCDR2, and HCDR3 shown in VH comprising the amino acid sequence of SEQ ID NO: 18, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence of LCDR1, LCDR2, and LCDR3 shown in VL comprising the amino acid sequence of SEQ ID NO: 19.

[0015] In some embodiments, the additional antigen-binding domain comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0016] In some embodiments, the additional antigen-binding domain comprises a VH domain and a VL domain, the VH domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 18, and the VL domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the additional antigen-binding domain comprises a VH domain and a VL domain, the VH domain comprising the amino acid sequence of SEQ ID NO: 18, and the VL domain comprising the amino acid sequence of SEQ ID NO: 19.

[0017] In some embodiments, the additional antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the additional antigen-binding domain comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 20.

[0018] In some embodiments, the antigen-binding domains are fused to each other via a peptide linker.

[0019] In some embodiments, the length of the peptide linker is about 50 amino acids or less.

[0020] In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α.

[0021] In some embodiments, the intracellular signaling domain includes the primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0022] In some embodiments, the intracellular signaling domain further includes a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of CD83, and combinations thereof. In some embodiments, the co-stimulatory signaling domain is derived from CD137.

[0023] In some embodiments, the CAR according to the present specification further includes a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α.

[0024] In some embodiments, the CAR according to the present specification further includes a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is from CD8α.

[0025] In some embodiments, the present specification provides a chimeric antigen receptor (CAR), which comprises (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 26 and SEQ ID NO: 28, or (ii) an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequences of SEQ ID NO: 26 and SEQ ID NO: 28.

[0026] In another aspect, the present specification provides a nucleic acid, which encodes an anti-claudin-6 sdAb, a fusion protein or a CAR according to the present specification, or a fragment thereof.

[0027] In another aspect, the nucleic acid according to the present specification further comprises a sequence encoding a chimeric receptor. In some embodiments, the nucleic acid encoding the chimeric receptor comprises the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid encoding the chimeric receptor encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 32 and 44-45.

[0028] In another aspect, the present specification provides a vector, which comprises the nucleic acid according to the present specification.

[0029] In another aspect, the present specification provides an engineered immune effector cell, which comprises a CAR, a nucleic acid and / or a vector according to the present specification. In some embodiments, the engineered immune effector cell is a T cell, an NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell or a combination thereof.

[0030] In another aspect, the present specification provides a pharmaceutical composition, which comprises an anti-claudin-6 sdAb, a nucleic acid, a vector or an engineered immune effector cell according to the present specification, and a pharmaceutically acceptable excipient.

[0031] In another aspect, the present specification provides a method for treating a target disease or disorder, the method comprising administering to the target an effective amount of an anti-Claudin-6 sdAb, engineered immune effector cells or pharmaceutical composition according to the present specification. In some aspects, the disease or disorder is a Claudin-6 related disease or disorder and / or a GPC3 related disease or disorder. In some aspects, the disease or disorder is cancer, such as solid tumor cancer. In some aspects, the disease or disorder is selected from the group consisting of germ cell tumor (GCT), ovarian cancer (OC), non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), endometrial cancer (EC) and AFP+ gastric cancer (AFP+GC).

[0032] In another aspect, the present disclosure further provides a method for treating a disease or disorder of a target in need thereof, the method comprising administering to the target an effective amount of a combination of a CLDN6 antagonist and a GPC3 antagonist, or an antagonist of CLDN6 and GPC3. The CLDN6 antagonist, GPC3 antagonist and / or antagonist of CLDN6 and GPC3 can have multiple classes. For example, the antagonist may be selected from engineered receptors, engineered immune cells, antibodies, antibody-drug conjugates (ADCs), aptamers and small interfering RNAs.

[0033] In one aspect, the CLDN6 antagonist is an engineered immune cell comprising an engineered receptor that specifically targets CLDN6, wherein the engineered receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that comprises at least one anti-CLDN6 binding moiety (e.g., an anti-CLDN6 sdAb according to the present specification). In some aspects, the GPC3 antagonist is an engineered immune cell comprising an engineered receptor that specifically targets GPC3, wherein the engineered receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that comprises at least one anti-GPC3 binding moiety. In some aspects, the engineered receptor that specifically targets CLDN6 (e.g., CLDN6 CAR according to the present specification) and the engineered receptor that specifically targets GPC3 are expressed in different engineered immune cells. Accordingly, the combination of the CLDN6 antagonist and the GPC3 antagonist is a combination of a first group of engineered immune cells comprising the engineered receptor that specifically targets CLDN6 described herein and a second group of engineered immune cells of the engineered receptor that specifically targets GPC3.

[0034] In some embodiments, the antagonists of CLDN6 and GPC3 are engineered immune cells that include a first engineered receptor that specifically targets CLDN6 and a second engineered receptor that specifically targets GPC3, i.e., the two engineered receptors are co-expressed in the same immune cell. More specifically, the antagonists of CLDN6 and GPC3 are engineered immune cells that include a first engineered receptor that specifically targets CLDN6 and a second engineered receptor that specifically targets GPC3, where (1) the first engineered receptor that specifically targets CLDN6 includes a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signaling domain that includes at least one anti-CLDN6 binding moiety, and (2) the second engineered receptor that specifically targets GPC3 includes a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signaling domain that includes at least one anti-GPC3 binding moiety.

[0035] In some embodiments, the antagonists of CLDN6 and GPC3 are engineered immune cells that include a first chimeric antigen receptor (CAR) that targets CLDN6 and a second CAR that targets GPC3, where (1) the first CAR includes a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signaling domain that contains at least one anti-CLDN6 binding moiety (e.g., an anti-CLDN6 sdAb according to the present specification), (2) the second CAR includes a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signaling domain that contains at least one anti-GPC3 binding moiety, where the first CAR and the second CAR are functionally linked, the first CAR is located at the N-terminus or C-terminus of the second CAR that targets GPC3, and optionally, the first CAR is functionally linked to the second CAR via a cleavable peptide, or the first CAR and the second CAR are not linked for cleavage of the cleavable peptide.

[0036] In some embodiments, the antagonists of CLDN6 and GPC3 are engineered immune cells comprising an engineered receptor that targets CLDN6 and GPC3 simultaneously, wherein the engineered receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least one anti-CLDN6 binding moiety (e.g., an anti-CLDN6 sdAb according to the present specification) and at least one anti-GPC3 binding moiety. In some embodiments, the antagonists of CLDN6 and GPC3 are engineered immune cells comprising a CAR that targets CLDN6 and GPC3 simultaneously (a "CLDN6×GPC3 bispecific CAR"), wherein the CLDN6×GPC3-specific CAR comprises (1) an extracellular antigen-binding domain comprising at least one anti-CLDN6 binding moiety and at least one anti-GPC3 binding moiety, (2) a transmembrane domain, and (3) an intracellular signaling domain, wherein the anti-CLDN6 binding moiety is located at the N-terminus or C-terminus of the anti-GPC3 binding moiety, and optionally, the anti-CLDN6 binding moiety is functionally linked to the anti-GPC3 binding moiety via a GS linker, e.g., a peptide linker such as (G4S)3.

[0037] In some embodiments, the disease or disorder is a claudin-6-related disease or disorder and / or a GPC3-related disease or disorder. In some embodiments, the disease or disorder is cancer, e.g., solid tumor cancer. In some embodiments, the disease or disorder is selected from the group consisting of germ cell tumor (GCT), ovarian cancer (OC), non-small cell lung cancer (NSCLC), hepatocellular cancer (HCC), endometrial cancer (EC), and AFP+ gastric cancer (AFP+GC). In some embodiments, the subject is resistant to at least one CLDN6 agent and / or wherein the subject is resistant to at least one GPC3 agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0038]

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Mode for Carrying Out the Invention

[0039] 5. Detailed Description The present disclosure is partially based on novel antibodies that bind to claudin-6, chimeric antigen receptors that bind to claudin-6 and / or GPC3, or engineered cells that express them and / or chimeric receptors, and their improved properties.

[0040] 5.1. Definitions The techniques and procedures described or referenced in this specification include, for example, widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (edited by Ausubel et al., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009), Monoclonal Antibodies: Methods and Protocols (edited by Albitar, 2010), and Antibody Engineering Volumes 1 and 2 (edited by Kontermann and Dubel, 2nd ed. 2010), which are generally well understood and / or commonly employed by those skilled in the art using conventional methods. Unless otherwise defined herein, technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. For the interpretation of this specification, the following explanations of terms apply, and in any appropriate case, terms used in the singular form also include the plural form, and vice versa. If any explanation of the terms described conflicts with any of the documents incorporated herein by reference, the explanation of the terms described below shall prevail.

[0041] The terms "antibody", "immunoglobulin", or "Ig" may be used interchangeably herein, are used in the broadest sense, and specifically include, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies as long as they exhibit the desired biological activity), single-chain antibodies and their fragments (e.g., domain antibodies), as described below. Antibodies can be human antibodies, humanized antibodies, chimeric antibodies and / or affinity matured antibodies as well as antibodies from other species (e.g., mouse, rabbit, llama, etc.). The term "antibody" is intended to include the polypeptide product of B cells in immunoglobulin-based polypeptides, which has the ability to bind to a specific molecular antigen and is composed of two identical polypeptide chain pairs, where each pair of polypeptide chains has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain contains a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain contains a constant region. See, for example, Antibodey Engineering (edited by Borrebaeck, 2nd edition 1995) and Kuby, Immunology (3rd edition 1997). Antibodies further include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, antibodies from camelid species (e.g., llama or alpaca) or their humanized variants, intrabodies, anti-idiotype (anti-Id) antibodies and any functional fragments (e.g., antigen-binding fragments) of the above, and these functional fragments refer to a part of the antibody heavy chain or light chain polypeptide and retain some or all of the binding activity of the antibody from which the fragment is derived.Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (including, for example, monospecific, bispecific, etc.), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, Fvs linked by disulfide bonds (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetra-bodies, and minibodies. Specifically, an antibody according to the present specification includes an immunoglobulin molecule and an immunologically active portion of the immunoglobulin molecule, for example, a molecule containing an antigen-binding domain or an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (edited by Myers, 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2nd Edition 1990). An antibody according to the present specification can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody can be an agonist antibody or an antagonist antibody. The antibody can be neither an agonist nor an antagonist.

[0042] An "antigen" is a structure to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. The target antigen can be a polypeptide. The antigen can be cell-associated, for example, present on or in a cell.

[0043] An "intact" antibody is an antibody that includes an antigen-binding site and CL and at least the heavy-chain constant regions CH1, CH2, and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. An intact antibody may have one or more effector functions.

[0044] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment that includes VH and VL antibody domains linked in a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker between the VH and VL domains, and the polypeptide linker enables the sFv to form a desirable structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315 (1994).

[0045] The term "heavy-chain only antibody" or "HCAb" includes a functional antibody that includes a heavy chain but lacks the light chain typically found in a four-chain antibody. For example, camelids (e.g., camels, llamas, or alpacas) are known to produce HCAb.

[0046] As used herein, the term "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain and is capable of binding to an antigen (e.g., a single domain antibody that binds to Claudin-6). Single domain antibodies include the VHH domains described herein. Examples of single domain antibodies include, but are not limited to, antibodies lacking a light chain naturally, such as antibodies from camelid species (e.g., llama), single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies (e.g., VHH domain antibodies) may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. For example, as described herein, single domain antibodies may be derived from antibodies produced from camelid species, such as camel, llama, dromedary, alpaca, and guanaco. Other species other than camelids are capable of producing heavy chain antibodies lacking a light chain naturally, and VHHs derived from such other species are within the scope of the present disclosure. In some embodiments, single domain antibodies (e.g., VHHs) according to the present specification have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein. Single domain antibodies can be part of a large binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0047] The term "binding" refers to an interaction between molecules and includes, for example, forming a complex. The interaction may be a non-covalent interaction including, for example, hydrogen bonding, ionic bonding, hydrophobic interaction and / or van der Waals interaction. The complex may further include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The overall strength of the non-covalent interaction between a single antigen binding site on an antibody and a single epitope of a target molecule (e.g., an antigen) is the affinity of the antibody or functional fragment for the epitope. The dissociation rate (k off ) of a binding molecule (e.g., an antibody) and a monovalent antigen and the association rate (kon ) and the ratio (k off / k on ) is the dissociation constant K D and is inversely proportional to the affinity. The lower the K D value, the higher the affinity of the antibody. The K D value varies depending on the complex of the antibody and the antigen and depends on k on and k off . The dissociation constant K D of the antibody according to this specification can be determined using any method according to this specification or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen (e.g., a multivalent antigen) containing multiple repeating antigenic determinants contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the likelihood of a reaction at the second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0048] In connection with the binding molecules described herein, terms such as "binds to", "specifically binds to", and similar terms are also used synonymously herein to refer to a binding molecule of an antigen-binding domain that specifically binds to an antigen (e.g., a polypeptide). A binding molecule or antigen-binding domain that binds or specifically binds to an antigen can be identified, for example, by immunoassay, Octet®, Biacore®, or other techniques well known to those of skill in the art. When a binding molecule or antigen-binding domain binds to an antigen with a higher affinity than any cross-reactive antigen, the binding molecule or antigen-binding domain can bind or specifically bind to the antigen as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective reaction will be at least 2-fold, and can be more than 10-fold, above the background signal or noise. See, for example, the discussion of binding specificity in Fundamental Immunology 332-36 (Paul, ed., 2nd ed. 1989). The degree to which a binding molecule or antigen-binding domain binds to a "non-target" protein can be, for example, about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, as determined by fluorescence-activated cell sorting (FACS) analysis or RIA. A binding molecule or antigen-binding domain that binds to an antigen includes a binding molecule or antigen-binding domain that can bind to the antigen with sufficient affinity such that, for example, the binding molecule can be used as a therapeutic and / or diagnostic agent that targets the antigen. A binding molecule or antigen-binding domain that binds to an antigen can have a dissociation constant (K D ) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. A binding molecule or antigen-binding domain can bind to an antigenic epitope that is conserved among antigens from different species.

[0049] The binding molecule or antigen-binding domain may contain a "chimeric" sequence, where a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass and fragments of such antibodies, provided they exhibit the desired biological activity (see U.S. Patent No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). The chimeric sequence may include a humanized sequence.

[0050] The binding molecule or antigen-binding domain may comprise a portion of a "humanized" form of a non-human (e.g., camelid, murine, non-human primate) antibody, where these non-human antibodies comprise sequences from human immunoglobulins (e.g., acceptor antibodies), and where the native CDR residues are replaced with residues from the corresponding CDRs of a non-human species (e.g., donor antibody) such as a camelid, mouse, rat, rabbit or non-human primate having the desired specificity, affinity and capacity. One or more FR region residues of the human immunoglobulin sequence may be replaced with the corresponding non-human residues. Additionally, the humanized antibody may comprise residues not found in the acceptor antibody or donor antibody. These modifications are made to further improve antibody performance. The humanized heavy or light chain may comprise substantially all of at least one or more variable regions, where all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin and all or substantially all of the FRs are the FRs of a human immunoglobulin sequence. The humanized antibody comprises at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of a human immunoglobulin constant region. For further details, see Jones et al., Nature 321:522-25 (1986), Riechmann et al., Nature 332:323-29 (1988), Presta, Curr. Op. Struct. Biol. 2:593-96 (1992), Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285-89 (1992), U.S. Pat. Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213 and 6,054,297.

[0051] The binding molecule or antigen-binding domain may include a part of a "fully human antibody" or "human antibody", where these terms are used synonymously herein and refer to an antibody comprising a human variable region and, for example, a human constant region. The binding molecule may include an antibody sequence. The term refers to an antibody that may include variable and constant regions derived from humans. A "fully human" antibody may further encompass an antibody that binds to a polypeptide and is encoded by a nucleic acid sequence that is a somatic variant of a naturally occurring human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences, for example, as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or produced using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991), Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68 (2006)). Methods that can also be used for the production of human monoclonal antibodies are described in the literature by Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985), Boerner et al., J. Immunol. 147(1):86-95 (1991), and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001).Human antibodies can be produced by administering an antigen to a transgenic animal (e.g., a mouse) whose endogenous locus has been inactivated but that has been modified to produce such antibodies in response to an antigen challenge (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995), Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997), and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). Human antibodies produced by human B cell hybridoma technology are further described, e.g., in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006).

[0052] The binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody", where the term includes antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from transgenic and / or translchromosomal animals (e.g., mice or cows) with respect to human immunoglobulin genes (see, e.g., Taylor, L. D. et al., Nucl. Acids Res. 20:6287-6295 (1992)) or antibodies produced, expressed, generated or isolated by any other method involving splicing to other DNA sequences of human immunoglobulin gene sequences, including human antibodies produced, expressed, generated or isolated by recombinant methods. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-XXX). However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, in the case of using transgenic animals with human Ig sequences, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that are derived from and closely related to human germline VH and VL sequences, but may be sequences that do not naturally occur within the human antibody germline repertoire in vivo.

[0053] The binding molecule or antigen-binding domain may comprise a portion of a "monoclonal antibody", where the term as used herein refers to an antibody obtained from a substantially homogeneous group of antibodies, e.g., each antibody constituting the group is identical except for possible naturally occurring mutations or well-known post-translational modifications that may be present in minor amounts (e.g., amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation), and each monoclonal antibody will typically recognize a single epitope on the antigen. As used herein, "monoclonal antibody" is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for producing the antibody. For example, monoclonal antibodies that may be used in the present disclosure may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be produced in bacterial or eukaryotic animal or plant cells using recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for producing clonal cell lines and the monoclonal antibodies they express are well known in the art. See, e.g., Short Protocols in Molecular Biology (edited by Ausubel et al., 5th ed. 2002).

[0054] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is usually about 150,000 daltons. Each L chain is linked to an H chain via one covalent disulfide bond, while the two H chains are linked to each other via one or more disulfide bonds depending on the H-chain isotype. Each H and L chain further has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, while four CH domains follow for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain (CL) at the other end. VL aligns with VH, and CL aligns with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form the interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. For the structural properties of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (edited by Stites et al., 8th ed. 1994) and Immunobiology (Janeway et al., 5th ed. 2001).

[0055] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. Conventional IgG typically contains two Fab regions, and each Fab region is present in one of the two arms of the Y-shaped IgG structure. Each Fab region is usually composed of one variable region and one constant region of each of the heavy and light chains. More specifically, the variable region and constant region of the heavy chain in the Fab region are the VH region and the CH1 region, respectively, and the variable region and constant region of the light chain in the Fab region are the VL region and the CL region, respectively. VH, CH1, VL, and CL in the Fab region can be arranged in various ways so as to confer the antigen-binding ability according to the present disclosure. For example, like the Fab region of conventional IgG, the VH region and the CH1 region may be on one polypeptide, and the VL region and the CL region may be on a single polypeptide. Alternatively, the VH, CH1, VL, and CL regions may all be on the same polypeptide and be oriented in different orders as described in more detail in the following sections.

[0056] The terms "variable region", "variable domain", "V region" or "V domain" refer to a part of the light or heavy chain of an antibody, which is usually located at the amino terminus of the light or heavy chain and is about 120-130 amino acids in length for the heavy chain and about 100-110 amino acids in length for the light chain, and is used for the binding and specificity of that particular antibody to its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that some segments of the variable regions in antibodies exhibit large differences in sequence. The V region mediates antigen binding and defines the specificity of that particular antibody to its particular antigen. However, the variability is not evenly distributed within the 110 amino acid range of the variable region. Instead, the V region is composed of segments of about 15-30 amino acids, called framework regions (FRs), which are less variable (e.g., relatively invariant), and these segments are separated by short regions, called "hypervariable regions", which are highly variable (e.g., extremely variable) and are about 9-12 amino acids in length each. The variable regions of the heavy and light chains each contain four FRs that mainly use a β-sheet structure, which are linked via three hypervariable regions. These three hypervariable regions are linked to form loops and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held very close together and held integrally by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibody (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0057] The terms "Kabat numbering of variable region residues" or "Kabat amino acid position numbering" and variations thereof refer to the numbering system used for the heavy chain variable region or light chain variable region of antibody sequences as described by Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or different amino acids corresponding to deletions or insertions in the variable domain FR or CDR. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues of a given antibody can be determined by alignment in the region of homology between the antibody sequence and the "standard" Kabat numbering sequence. When referring to residues in the variable domain (approximately residues 1 - 107 of the light chain and residues 1 - 113 of the heavy chain), the Kabat numbering system (e.g., as described by Kabat et al., supra) is usually used. When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" (e.g., the EU index reported by Kabat et al., supra) is usually used. The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0058] When used in connection with an antibody, the term "heavy chain" refers to a polypeptide chain of about 50 - 70 kDa, wherein the amino-terminal portion contains a variable region of about 120 - 130 or more amino acids and the carboxy-terminal portion contains a constant region. The amino acid sequence based on the heavy chain constant region can be one of five different types (e.g., isotypes) designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Separate heavy chains differ in size: α, δ, and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, these different types of heavy chains each produce one of the five well-known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

[0059] When used in connection with an antibody, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion contains a variable region of about 100 - about 110 or more amino acids and the carboxy-terminal portion contains a constant region. The approximate length of the light chain is 211 - 217 amino acids. There are two different types of amino acid sequences based on the constant domain, designated kappa (κ) or lambda (λ).

[0060] As used herein, the terms "hypervariable region", "HVR", "complementary determining region", and "CDR" are used synonymously. "CDR" refers to one of three hypervariable regions (H1, H2, or H3) within the non-framework regions of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of three hypervariable regions (L1, L2, or L3) within the non-framework regions of the VL β-sheet framework of an antibody. CDR1, CDR2, and CDR3 in the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively. CDR1, CDR2, and CDR3 in the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Thus, a CDR is a variable region sequence that is interspersed within the range of the framework region sequences.

[0061] The CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, the Kabat Complementary Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra, Nick Deschacht et al., J Immunol 2010; 184:5696-5704). Chothia, instead, refers to the positions of structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When numbered using the Kabat numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34, which depends on the length of the loop (this is because the Kabat numbering regimen places insertions at H35A and H35B, and if neither 35A nor 35B is present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions correspond to a compromise between the Kabat CDRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Volume 2 (edited by Kontermann and Dubel, 2nd Edition, 2010)). The "Contact" hypervariable regions are based on analysis of available complex crystal structures. Another widely adopted and general-purpose numbering system that has been developed is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system specialized for human and other vertebrate immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs). As used herein, CDRs refer to the amino acid sequences and positions within the light or heavy chains. The "positions" of the CDRs within the structure of the immunoglobulin variable domain are conserved across species and are present within structures called loops, so that CDRs and framework residues can be readily identified by using a numbering system that aligns the variable domain sequences according to structural features.This information can be used to graft and replace CDR residues from one species' immunoglobulin onto an acceptor framework, usually from a human antibody. Honegger and Pluckthun, J. Mol. Biol. 309: 657-70 (2001), developed an alternative numbering system (AHon). Correspondence between numbering systems, including, for example, Kabat numbering and the IMGT proprietary numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from these hypervariable regions or CDRs are illustrated in Table 1 below.

[0062] [Table 1]

[0063] The boundaries of a given CDR can vary depending on the regime used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or region thereof (e.g., variable region), and each CDR of the antibody or region thereof (e.g., CDR-H1, CDR-H2), are to be understood to encompass the complementary determining regions defined by any of the above-known regimes. In some cases, a regime for identifying one or more specific CDRs is specified, e.g., the CDRs defined by the IMGT, Kabat, Chothia, or Contact methods. In other cases, a specific amino acid sequence of the CDR is given. It should be noted that the CDR regions may be defined by combinations of various numbering systems, e.g., combinations of the Kabat and Chothia numbering systems, combinations of the Kabat and AbM numbering systems, or combinations of the Kabat and IMGT numbering systems. Thus, terms such as "CDR1 as described in a particular VH" include, but are not limited to, any CDR1 defined by the above-exemplified CDR numbering systems. Given a variable region (e.g., VH or VL), one of ordinary skill in the art will understand that the CDRs within that region can be defined by various numbering systems or combinations thereof.

[0064] The hypervariable regions may include "extended hypervariable regions" such as 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0065] The terms "constant region" or "constant domain" refer to the carboxy-terminal portions of the light and heavy chains, which do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions, such as interaction with Fc receptors. The term refers to a part of the immunoglobulin molecule, and this part of the immunoglobulin molecule has a more conserved amino acid sequence compared to other parts of the immunoglobulin, i.e., the variable region, and the amino acid sequence contains the antigen-binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0066] The terms "framework" or "FR" refer to variable region residues located on both sides of the CDRs. FR residues are present, for example, in chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region residues or CDR residues.

[0067] The term "Fc region" as used herein is for defining the C-terminal region of an immunoglobulin heavy chain and includes, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxy terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include a group of antibodies in which all K447 residues have been removed, a group of antibodies in which the K447 residues have not been removed, and a group of antibodies having a mixture of antibodies containing the K447 residue and those not containing it. A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally need to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) of the Fc region and can be evaluated using various assays well known to those skilled in the art. A "variant Fc region" includes an amino acid sequence that is different from the native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion). In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parental polypeptide, for example, having about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of a parental polypeptide. The variant Fc regions herein may have at least about 80% homology, or at least about 90% homology, or for example, at least about 95% homology with the native sequence Fc region and / or the Fc region of a parental polypeptide.

[0068] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope may be a linear epitope or a conformational, non-linear or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope may be a continuous amino acid of the polypeptide ("linear" epitope), or an epitope may include amino acids from two or more non-contiguous regions of the polypeptide ("conformational", "non-linear" or "discontinuous" epitope). Those skilled in the art will generally understand that linear epitopes may or may not depend on secondary, tertiary or quaternary structure. For example, a binding molecule can bind to a group of amino acids regardless of whether it is folded into a native three-dimensional protein structure. In order for a binding molecule to recognize and bind an epitope, the amino acid residues constituting the epitope need to exhibit a specific conformation (e.g., bend, twist, turn or fold).

[0069] "Percent (%) sequence identity" and "homology" of a peptide, polypeptide or antibody sequence are defined as the percent of amino acid residues in a candidate sequence that are the same as the amino acid residues in a particular peptide or polypeptide sequence when sequence alignment is performed to achieve the maximum percent sequence identity and gaps are introduced if necessary, and no conservative substitutions are considered part of the sequence identity. Alignment can be achieved and the percent amino acid sequence identity determined using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software in various ways within the skill of the art. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

[0070] The term "specificity" refers to the selective discrimination of a particular epitope of an antigen by an antigen-binding protein (e.g., a CAR or an antibody). For example, a natural antibody is monospecific. As used herein, the term "multispecificity" indicates that an antigen-binding protein (e.g., a CAR or an antibody) has two or more antigen-binding sites, where at least two bind to different antigens. As used herein, "bispecificity" indicates that an antigen-binding protein (e.g., a CAR or an antibody) has two different antigen-binding specificities. As used herein, a "monospecific" CAR refers to an antigen-binding protein (e.g., a CAR or an antibody) having one or more binding sites, where each binding site binds to the same antigen.

[0071] As used herein, the term "valence" indicates that a given number of binding sites are present on an antigen-binding protein (e.g., a CAR or an antibody). For example, a natural or full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" indicate that two, three, four, five, and six binding sites, respectively, are present on an antigen-binding protein (e.g., a CAR or an antibody).

[0072] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically graft one or more antigens onto immune effector cells such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." A CAR may include an extracellular antigen-binding domain specific for one or more antigens (e.g., tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T cell" refers to a T cell that expresses a CAR.

[0073] The terms "polypeptide", "peptide", and "protein" are used synonymously herein and refer to polymers of amino acids of any length. The polymers may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acids. These terms further encompass amino acid polymers that have been naturally modified or modified by intervention. For example, formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition further includes, for example, polypeptides containing one or more amino acid analogs, including, but not limited to, unnatural amino acids and other modifications known in the art. It should be understood that since the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, for example, a "polypeptide" may exist as a single-stranded or two or more associated strands.

[0074] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or a synthetic reaction. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, usually single-stranded, synthetic polynucleotide, the length of which is usually (but not necessarily) less than about 200 nucleotides. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells producing the binding molecules of the present disclosure may include parental hybridoma cells, as well as bacteria and eukaryotic host cells into which nucleic acids encoding antibodies have been introduced. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5'-end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of addition of a nascent RNA transcript from 5' to 3' is referred to as the transcription direction. A sequence region on the 5'-side of the 5'-end of an RNA transcript that has the same sequence as the RNA transcript in a DNA strand is referred to as an "upstream sequence", and a sequence region on the 3'-side of the 3'-end of an RNA transcript that has the same sequence as the RNA transcript in a DNA strand is referred to as a "downstream sequence".

[0075] "Isolated nucleic acid" refers to a nucleic acid (e.g., RNA, DNA, or a mixture of nucleic acids) that is substantially separated from other genomic DNA sequences that are naturally associated with the native sequence, as well as proteins or complexes such as ribosomes and polymerases. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Note that an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may not substantially contain other cellular material or medium when produced by recombinant techniques, or may not substantially contain chemical precursors or other chemicals when chemically synthesized. One or more nucleic acid molecules encoding the antibodies described herein can be isolated or purified. The term includes nucleic acid sequences removed from their natural environment, and includes recombinant or cloned DNA isolates and biologically synthesized analogs by chemically synthesized analogs or heterologous systems. A substantially pure molecule may include the isolated form of the molecule. Specifically, the "isolated" nucleic acid molecule encoding a CAR or antibody described herein is a nucleic acid molecule that is identified and isolated from at least one contaminating nucleic acid molecule, which nucleic acid molecule is normally associated with the at least one contaminating nucleic acid molecule in the environment in which it is produced.

[0076] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are in degenerate forms to each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may further include introns to the extent that the nucleotide sequence encoding the protein may contain one or more introns in some forms.

[0077] The term "control sequence" refers to the DNA sequences necessary to express a functionally linked coding sequence in a particular host organism. Control sequences applicable to prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize a promoter, a polyadenylation signal, and an enhancer.

[0078] As used herein, the term "functionally linked" and similar phrases (e.g., genetically fused), when used to refer to nucleic acids or amino acids, respectively refer to an operable linkage of nucleic acid sequences or amino acid sequences, which are placed in a functional relationship with each other. For example, when a promoter, enhancer element, open reading frame, 5' and 3' UTRs, and terminator sequences are operably linked, accurate production of a nucleic acid molecule (e.g., RNA) occurs. Functionally linked nucleic acid elements can cause transcription of an open reading frame and ultimately cause production of a polypeptide (i.e., expression of the open reading frame). As another example, operably linked peptides are peptides in which the functional domains are placed at an appropriate distance from each other and confer the desired function on each domain.

[0079] The term "vector" refers to a substance for carrying or containing a nucleic acid sequence, such as a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, for introducing the nucleic acid sequence into a host cell. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which may include a selection sequence or a label that can be stably integrated into the host cell chromosome. Note that the vector may include one or more selectable marker genes and appropriate expression control sequences. The selectable marker genes included provide, for example, resistance to antibiotics or toxins, supplement nutritional deficiencies or supply essential nutrients not present in the medium. The expression control sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, etc. well known in the art. When attempting to co-express two or more nucleic acid molecules (e.g., antibody heavy and light chains or antibody VH and VL), the two nucleic acid molecules may be inserted, for example, into a single expression vector or separate expression vectors. For single vector expression, the coding nucleic acids may be operably linked to one common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. These methods include, for example, nucleic acid analysis such as Northern blotting of mRNA or polymerase chain reaction (PCR) amplification, immunoblotting used for the expression of gene products or other appropriate analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art should understand that the nucleic acid molecules are expressed in an amount sufficient to produce the desired product, and further should understand that the expression levels can be optimized by methods well known in the art to obtain sufficient expression.

[0080] As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

[0081] As used herein, the term "host cell" refers to a particular target cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such cells. The progeny of such cells may not be the same as the parental cells transfected with the nucleic acid molecule due to mutations that may occur in subsequent generations, environmental influences, or integration of the nucleic acid molecule into the host cell genome.

[0082] As used herein, the term "autologous" refers to any material derived from the same individual, where the material is later reintroduced into the individual.

[0083] "Allogeneic" refers to a graft derived from different individuals of the same species.

[0084] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing foreign nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. The cells include the primary target cells and their progeny.

[0085] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the U.S. Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopeias for use in animals and more specifically in humans.

[0086] "Excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, adhesives, buffers, carrier agents, coating agents, coloring agents, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, release agents, sterilizing agents, sweetening agents, solubilizing agents, wetting agents, and mixtures thereof. The term "excipient" may further refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0087] The excipient may be a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates and other organic acids, antioxidants including ascorbic acid, polypeptides of low molecular weight (e.g., less than about 10 amino acid residues), proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th Edition, 1990).

[0088] Each component may be "pharmaceutically acceptable" in the sense that it is compatible with the other components of the pharmaceutical formulation and, in proportion to a reasonable risk-benefit ratio, is suitable for contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity or other problems or complications. See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th Edition; Edited by Rowe et al., The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd Edition, Edited by Ash and Ash, Gower Publishing Company: 2007, Pharmaceutical Preformulation and Formulation, 2nd Edition, Edited by Gibson, CRC Press LLC: Boca Raton, FL, 2009. Pharmaceutically acceptable excipients can be non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations used. Pharmaceutically acceptable excipients may be aqueous pH buffer solutions.

[0089] Excipients may be sterile liquids such as oils and water, including those from petroleum, animal, vegetable or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary excipient. Aqueous saline solutions, aqueous glucose solutions and glycerin solutions can also be used as liquid excipients, especially for injection solutions. Excipients may further include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, non-fat dry milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. The composition may further include a small amount of wetting agent or emulsifying agent or pH buffering agent as needed. The composition can take forms such as solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions include formulations and may also include standard excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0090] Compositions containing a drug compound may include, for example, a binding molecule (e.g., an antibody) in isolated or purified form and a suitable amount of excipient.

[0091] As used herein, the terms "effective amount" or "therapeutically effective amount" refer to an amount sufficient to produce a desired result of an antibody or agent and a therapeutic molecule including the antibody or pharmaceutical composition according to this specification.

[0092] The terms "subject" and "patient" may be used synonymously. A subject as used herein may be a mammal such as a non-primate or a primate (e.g., a human). The subject may be a human. The subject may be a mammal diagnosed with a disease or disorder, e.g., a human. Alternatively, the subject may be a mammal at risk of developing a disease or disorder, e.g., a human.

[0093] "Administration" refers to the act of injecting or otherwise physically delivering a substance existing outside the body into the body of a patient by, for example, mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or well-known in the art.

[0094] As used herein, the terms "treat", "treatment", and "treating" refer to reducing or ameliorating the progression, severity, and / or duration of a disease or condition by administration of one or more therapies. Treatment can be determined by evaluating whether one or more symptoms associated with a latent condition are reduced, in remission, and / or alleviated such that improvement in the patient is observed, even though the patient may still have the latent condition. The term "treating" includes the control and amelioration of a disease. The terms "manage", "managing", and "management" refer to a beneficial effect obtained from a therapy that does not necessarily result in a cure of the disease.

[0095] The term "antagonist" refers to a substance that interferes with or inhibits the physiological action of a target antigen or a target antigen-mediated signaling pathway. Various types of antagonists are known in the art and include, but are not limited to, engineered immune cells, engineered receptors such as engineered T cell receptors (TCRs), chimeric antigen receptors (CARs), T cell antigen coupling agents (TACs) or portions thereof, antibodies, antibody-drug conjugates (ADCs), aptamers, small interfering RNAs, and chemical compound inhibitors.

[0096] As used herein, the term "combination of a CLDN6 antagonist and a GPC3 antagonist" refers to a combination of a first substance that specifically antagonizes anti-CLDN6 but does not antagonize GPC3 and a second substance that specifically antagonizes GPC3 but does not antagonize CLDN6. The first substance and the second substance may be separated or may be linked, fused, or conjugated. The CLDN6 antagonist may be a first CAR having an extracellular antigen-binding domain that binds to CLDN6 but not to GPC3, and the GPC3 antagonist may be a second CAR having an extracellular antigen-binding domain that binds to GPC3 but not to CLDN6. The first CAR and the second CAR may be individual polypeptides or may be linked together on the same chain. The CLDN6 antagonist may be a first group of engineered immune cells that express a CAR that binds to CLDN6 but not to GPC3, and the GPC3 antagonist may be a second group of engineered immune cells that express a CAR that binds to GPC3 but not to CLDN6. When the first substance and the second substance can be linked or fused as a new molecule that retains the antagonistic ability of both substances, the new molecule may be regarded as an "antagonist of CLDN6 and GPC3".

[0097] As used herein, the term "antagonist of CLDN6 and GPC3" refers to a substance having the ability to antagonize both CLDN6 and GPC3. The antagonist of CLDN6 and GPC3 may be a CAR having an extracellular antigen-binding domain that can bind to both CLDN6 and GPC3. The antagonist of CLDN6 and GPC3 may be an engineered immune cell that expresses a CAR having an extracellular antigen-binding domain that can bind to both CLDN6 and GPC3. The antagonist of CLDN6 and GPC3 may be an engineered immune cell that expresses a first CAR and a second CAR. The first CAR has an extracellular antigen-binding domain that binds to CLDN6 but not to GPC3, and the second CAR has an extracellular antigen-binding domain that binds to GPC3 but not to CLDN6. The antagonist of CLDN6 and GPC3 may also be an anti-CLDN6×GPC3 antibody.

[0098] The terms "prevent", "preventing", and "prevention" refer to reducing the likelihood that a disease, disorder, condition, or related symptom (e.g., diabetes or cancer) will occur (or recur).

[0099] As used herein, "delaying" the progression of cancer refers to retarding, suppressing, decelerating, extending, stabilizing, and / or slowing the progression of the disease. This delay may have different durations depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, since the individual is not suffering from the disease, a sufficient or significant delay may, in practice, include prevention. A method of "delaying" the progression of cancer, compared to not using the method, is a method that reduces the probability of disease progression within a given time frame and / or reduces the extent of the disease within a given time frame. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. The progression of cancer can be detected using standard methods, which include, but are not limited to, computed axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Progression may refer to the progression of cancer that may initially be undetectable and includes occurrence, recurrence, and onset.

[0100] As used herein, "claudin-6 related disease or disorder" refers to a disease or disorder that includes cells or tissues in which claudin-6 is expressed or abnormally expressed (e.g., overexpressed). A claudin-6 related disease or disorder may include cells in which claudin-6 is abnormally expressed. A claudin-6 related disease or disorder may include cells in which at least one activity of claudin-6 is lacking therein or therein. In some embodiments, the claudin-6 related disease or disorder is cancer, such as solid tumor cancer.

[0101] As used herein, the term "GPC3-related disease or disorder" refers to a disease or disorder that includes cells or tissues in which GPC3 is expressed or overexpressed. A GPC3-related disease or disorder may include cells in which GPC3 is abnormally expressed. A GPC3-related disease or disorder may include cells in which at least one activity of GPC3 is lacking therein or therein. In some embodiments, the GPC3-related disease or disorder is cancer, such as solid tumor cancer.

[0102] The terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1% or less of a given value or range.

[0103] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise.

[0104] It should be understood that aspects are described herein using the term "comprising" and similar aspects described using "consisting of" and / or "consisting essentially of" are also provided. It should be further understood that aspects are described herein using the phrase "consisting essentially of" and similar aspects described using "consisting of" are also provided.

[0105] The term "between" as used in the phrase "between A and B" or "between A~B" refers to a range that includes A and B.

[0106] The term "and / or" as used in phrases such as "A and / or B" herein is intended to include A and B, A or B, A alone, and B alone. Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the aspects of A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0107] 5.2. Single-domain antibody 5.2.1. Single-domain antibody that binds to Claudin-6 In one aspect, the present specification provides a single-domain antibody (e.g., VHH domain) that can bind to Claudin-6.

[0108] In some aspects, the single-domain antibody (e.g., VHH domain) according to the present specification binds to human Claudin-6. CLDN6 (UniProtKB:P56747) is a four-transmembrane protein involved in the formation of tight junctions during development.

[0109] The anti-Claudin-6 single-domain antibody according to the present specification can regulate the activity of one or more Claudin-6. In some aspects, the anti-Claudin-6 single-domain antibody according to the present specification is an antagonist antibody.

[0110] The anti-Claudin-6 single-domain antibody according to the present specification has a dissociation constant (K -8 of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -13 M to 10 -9 M, e.g., 10 -13 M to 10 D) can bind to Claudin-6 (e.g., human Claudin-6). Multiple methods for measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present disclosure, such as those by RIA using, for example, the Fab form of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81), those by biolayer interferometry (BLI) or surface plasmon resonance (SPR) methods, those by Octet® using, for example, the Octet® Red96 system, or those by Biacore® using, for example, Biacore® TM-2000 or Biacore® TM-3000. The "association rate" or "kon" can also be measured by the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques as above, using, for example, the Octet® Red96, Biacore® TM-2000 or Biacore® TM-3000 systems.

[0111] In some embodiments, the anti-Claudin-6 single domain antibody according to the present specification is a VHH domain. The exemplary VHH domains according to the present specification are produced as described in the following Section 6, and these VHH domains are designated 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1, and 77LICNB02H2.

[0112] Therefore, in some embodiments, the single domain antibody according to the present specification comprises one or more CDR sequences of any one of 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1, and 77LICNB02H2. In some embodiments, the present specification provides a single domain antibody that binds to Claudin-6 and comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences are selected from those in 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1, and 77LICNB02H2.

[0113] In some embodiments, provided are anti-claudin-6 single domain antibodies comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO:7. In some embodiments, provided are anti-claudin-6 single domain antibodies comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO:8. In some embodiments, provided are anti-claudin-6 single domain antibodies comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO:9. In some embodiments, provided are anti-claudin-6 single domain antibodies comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO:10. In some embodiments, provided are anti-claudin-6 single domain antibodies comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO:11. In some embodiments, the anti-claudin-6 single domain antibody is from a camelid. In some embodiments, the anti-claudin-6 single domain antibody is humanized. The anti-claudin-6 single domain antibody may comprise a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0114] In some embodiments, the single-domain antibody has a CDR1, and the CDR1 has the amino acid sequence of CDR1 shown in SEQ ID NO:7. In some embodiments, the single-domain antibody has a CDR2, and the CDR2 has the amino acid sequence of CDR2 shown in SEQ ID NO:7. In other embodiments, the single-domain antibody has a CDR3, and the CDR3 has the amino acid sequence of CDR3 shown in SEQ ID NO:7. In some embodiments, the single-domain antibody has CDR1 and CDR2, and the CDR1 and CDR2 have the amino acid sequences of CDR1 and CDR2 shown in SEQ ID NO:7. In some embodiments, the single-domain antibody has CDR1 and CDR3, and the CDR1 and CDR3 have the amino acid sequences of CDR1 and CDR3 shown in SEQ ID NO:7. In some embodiments, the single-domain antibody has CDR2 and CDR3, and the CDR2 and CDR3 have the amino acid sequences of CDR2 and CDR3 shown in SEQ ID NO:7. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:7. The CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are determined according to the IMGT numbering scheme. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the AbM numbering scheme. In other embodiments, the CDRs are determined according to the Chothia numbering scheme. In other embodiments, the CDRs are determined according to the Contact numbering scheme. The CDRs can be determined according to any combination of the above numbering schemes. In some embodiments, the anti-claudin-6 single-domain antibody is from a camelid. In some embodiments, the anti-claudin-6 single-domain antibody is humanized. The anti-claudin-6 single-domain antibody may include a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0115] In some embodiments, the single-domain antibody has a CDR1, and the CDR1 has the amino acid sequence of CDR1 shown in SEQ ID NO:8. In some embodiments, the single-domain antibody has a CDR2, and the CDR2 has the amino acid sequence of CDR2 shown in SEQ ID NO:8. In other embodiments, the single-domain antibody has a CDR3, and the CDR3 has the amino acid sequence of CDR3 shown in SEQ ID NO:8. In some embodiments, the single-domain antibody has CDR1 and CDR2, and the CDR1 and CDR2 have the amino acid sequences of CDR1 and CDR2 shown in SEQ ID NO:8. In some embodiments, the single-domain antibody has CDR1 and CDR3, and the CDR1 and CDR3 have the amino acid sequences of CDR1 and CDR3 shown in SEQ ID NO:8. In some embodiments, the single-domain antibody has CDR2 and CDR3, and the CDR2 and CDR3 have the amino acid sequences of CDR2 and CDR3 shown in SEQ ID NO:8. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:8. The CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are determined according to the IMGT numbering scheme. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the AbM numbering scheme. In other embodiments, the CDRs are determined according to the Chothia numbering scheme. In other embodiments, the CDRs are determined according to the Contact numbering scheme. The CDRs can be determined according to any combination of the above numbering schemes. In some embodiments, the anti-claudin-6 single-domain antibody is from a camelid. In some embodiments, the anti-claudin-6 single-domain antibody is humanized. The anti-claudin-6 single-domain antibody includes a recipient human framework, for example, a human immunoglobulin framework or a human consensus framework.

[0116] In some embodiments, the single-domain antibody has a CDR1, and the CDR1 has the amino acid sequence of CDR1 shown in SEQ ID NO:9. In some embodiments, the single-domain antibody has a CDR2, and the CDR2 has the amino acid sequence of CDR2 shown in SEQ ID NO:9. In other embodiments, the single-domain antibody has a CDR3, and the CDR3 has the amino acid sequence of CDR3 shown in SEQ ID NO:9. In some embodiments, the single-domain antibody has CDR1 and CDR2, and the CDR1 and CDR2 have the amino acid sequences of CDR1 and CDR2 shown in SEQ ID NO:9. In some embodiments, the single-domain antibody has CDR1 and CDR3, and the CDR1 and CDR3 have the amino acid sequences of CDR1 and CDR3 shown in SEQ ID NO:9. In some embodiments, the single-domain antibody has CDR2 and CDR3, and the CDR2 and CDR3 have the amino acid sequences of CDR2 and CDR3 shown in SEQ ID NO:9. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:9. CDR sequences can be determined according to well-known numbering systems. In some embodiments, CDRs are determined according to the IMGT numbering scheme. In some embodiments, CDRs are determined according to the Kabat numbering scheme. In some embodiments, CDRs are determined according to the AbM numbering scheme. In other embodiments, CDRs are determined according to the Chothia numbering scheme. In other embodiments, CDRs are determined according to the Contact numbering scheme. CDRs can be determined according to any combination of the above numbering schemes. In some embodiments, the anti-claudin-6 single-domain antibody is from a camelid. In some embodiments, the anti-claudin-6 single-domain antibody is humanized. The anti-claudin-6 single-domain antibody includes a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0117] In some embodiments, the single-domain antibody has CDR1, and the CDR1 has the amino acid sequence of CDR1 shown in SEQ ID NO:10. In some embodiments, the single-domain antibody has CDR2, and the CDR2 has the amino acid sequence of CDR2 shown in SEQ ID NO:10. In other embodiments, the single-domain antibody has CDR3, and the CDR3 has the amino acid sequence of CDR3 shown in SEQ ID NO:10. In some embodiments, the single-domain antibody has CDR1 and CDR2, and the CDR1 and CDR2 have the amino acid sequences of CDR1 and CDR2 shown in SEQ ID NO:10. In some embodiments, the single-domain antibody has CDR1 and CDR3, and the CDR1 and CDR3 have the amino acid sequences of CDR1 and CDR3 shown in SEQ ID NO:10. In some embodiments, the single-domain antibody has CDR2 and CDR3, and the CDR2 and CDR3 have the amino acid sequences of CDR2 and CDR3 shown in SEQ ID NO:10. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:10. The CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are determined according to the IMGT numbering scheme. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the AbM numbering scheme. In other embodiments, the CDRs are determined according to the Chothia numbering scheme. The CDRs can be determined according to any combination of the above numbering schemes. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the anti-claudin-6 single-domain antibody is from a camelid. In some embodiments, the anti-claudin-6 single-domain antibody is humanized. The anti-claudin-6 single-domain antibody includes a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0118] In some embodiments, the single-domain antibody has a CDR1, and the CDR1 has the amino acid sequence of CDR1 shown in SEQ ID NO:11. In some embodiments, the single-domain antibody has a CDR2, and the CDR2 has the amino acid sequence of CDR2 shown in SEQ ID NO:11. In other embodiments, the single-domain antibody has a CDR3, and the CDR3 has the amino acid sequence of CDR3 shown in SEQ ID NO:11. In some embodiments, the single-domain antibody has CDR1 and CDR2, and the CDR1 and CDR2 have the amino acid sequences of CDR1 and CDR2 shown in SEQ ID NO:11. In some embodiments, the single-domain antibody has CDR1 and CDR3, and the CDR1 and CDR3 have the amino acid sequences of CDR1 and CDR3 shown in SEQ ID NO:11. In some embodiments, the single-domain antibody has CDR2 and CDR3, and the CDR2 and CDR3 have the amino acid sequences of CDR2 and CDR3 shown in SEQ ID NO:11. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:11. The CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are determined according to the IMGT numbering scheme. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the AbM numbering scheme. In other embodiments, the CDRs are determined according to the Chothia numbering scheme. In other embodiments, the CDRs are determined according to the Contact numbering scheme. The CDRs can be determined according to any combination of the above numbering schemes. In some embodiments, the anti-claudin-6 single-domain antibody is from a camelid. In some embodiments, the anti-claudin-6 single-domain antibody is humanized. The anti-claudin-6 single-domain antibody includes a recipient human framework, for example, a human immunoglobulin framework or a human consensus framework.

[0119] In some embodiments, the present specification provides a single domain antibody that binds to Claudin-6 and includes a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where (i) CDR1 includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, (ii) CDR2 includes the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, and / or (iii) CDR3 includes the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the anti-Claudin-6 single domain antibody is from a camelid. In some embodiments, the anti-Claudin-6 single domain antibody is humanized. The anti-Claudin-6 single domain antibody may include a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0120] In other aspects, the present specification provides a single-domain antibody that binds to Claudin-6 and includes a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where (i) CDR1 includes an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, (ii) CDR2 includes an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:3 or SEQ ID NO:4, and / or (iii) CDR3 includes an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:5 or SEQ ID NO:6. In some aspects, the anti-Claudin-6 single-domain antibody is from a camelid. In some aspects, the anti-Claudin-6 single-domain antibody is humanized. The anti-Claudin-6 single-domain antibody may include a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0121] In some aspects, CDR1 includes the amino acid sequence of SEQ ID NO:1, CDR2 includes the amino acid sequence of SEQ ID NO:3, and CDR3 includes the amino acid sequence of SEQ ID NO:5. In some aspects, the anti-Claudin-6 single-domain antibody is from a camelid. In some aspects, the anti-Claudin-6 single-domain antibody is humanized. The anti-Claudin-6 single-domain antibody may include a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0122] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:2, CDR2 comprises the amino acid sequence of SEQ ID NO:4, and CDR3 comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the anti-claudin-6 single domain antibody is from a camelid. In some embodiments, the anti-claudin-6 single domain antibody is humanized. The anti-claudin-6 single domain antibody may comprise a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0123] In some embodiments, the single domain antibody further comprises one or more framework regions of 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1, and / or 77LICNB02H2. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO:7. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO:8. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO:9. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO:10. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO:11.

[0124] In some embodiments, the single domain antibody according to the present specification is a humanized single domain antibody. The humanized single domain antibody can be generated using the methods exemplified in Section 6 below or the methods described in the following sections.

[0125] The framework regions described herein are determined according to the boundaries of the CDR numbering system. In other words, when the CDRs are determined, for example, by Kabat, IMGT or Chothia, the framework regions are the amino acid residues that surround the CDRs in the form from the N-terminus to the C-terminus in the variable region: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residue at the N-terminus of the CDR1 amino acid residues defined by, for example, the Kabat numbering system, the AbM numbering system, the IMGT numbering system or the Chothia numbering system, FR2 is defined as the amino acid residue between the CDR1 and CDR2 amino acid residues defined by, for example, the Kabat numbering system, the AbM numbering system, the IMGT numbering system or the Chothia numbering system, FR3 is defined as the amino acid residue between the CDR2 and CDR3 amino acid residues defined by, for example, the Kabat numbering system, the AbM numbering system, the IMGT numbering system or the Chothia numbering system, and FR4 is defined as the amino acid residue at the C-terminus of the CDR3 amino acid residues defined by, for example, the Kabat numbering system, the AbM numbering system, the IMGT numbering system or the Chothia numbering system.

[0126] In some embodiments, provided is an isolated anti-claudin-6 single domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO:7. In some embodiments, provided is a polypeptide comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, provided is an isolated anti-claudin-6 single domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO:8. In some embodiments, provided is a polypeptide comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, provided is an isolated anti-claudin-6 single domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO:9. In some embodiments, provided is a polypeptide comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, provided is an isolated anti-claudin-6 single domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO:10. In some embodiments, provided is a polypeptide comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, provided is an isolated anti-claudin-6 single domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO:11. In some embodiments, provided is a polypeptide comprising the amino acid sequence of SEQ ID NO:11.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof described herein comprises an amino acid sequence having a certain percent identity to any one of antibodies 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1, and 77LICNB02H2.

[0128] The percent identity between two sequences, such as amino acid sequences or nucleic acid sequences, can be determined using a mathematical algorithm. Non-limiting examples of mathematical algorithms for comparing two sequences are the algorithms of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 87:2264–2268 (1990), which were modified in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873–5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, for example, a BLAST nucleotide search can be performed using the NBLAST nucleotide program parameter set with score = 100 and wordlength = 12. To obtain amino acid sequences homologous to the protein molecules described herein, for example, a BLAST protein search can be performed using the XBLAST program parameter set with score 50 and wordlength = 3. Gapped BLAST, described in Altschul et al., Nucleic Acids Res. 25:3389–3402 (1997), can be used to obtain gapped alignments for comparison purposes. Alternatively, PSI BLAST can be used for iterative searches (Id.) to detect distant relationships between molecules. When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., for XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) of the World Wide Web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS 4:11–17 (1998). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When comparing amino acid sequences using the ALIGN program, the PAM120 weight residue table can be used, the gap length penalty is 12, and the gap penalty is 4. Whether gaps are allowed or not, the percent identity between two sequences can be determined using a technique similar to the above technique. When calculating percent identity, usually only exact matches are counted.

[0129] In some embodiments, provided is an anti-claudin-6 single domain antibody comprising a VHH domain, wherein the VHH domain has at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NOs: 7-11. In some embodiments, any one of the VHH sequences having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity includes substitutions (e.g., conservative substitutions), insertions or deletions with respect to the reference sequence, but the anti-claudin-6 single domain antibody comprising the sequence retains the ability to bind to claudin-6. In some embodiments, in the amino acid sequence selected from SEQ ID NOs: 7-11, a total of 1-10 amino acids are substituted, inserted and / or deleted. In some embodiments, the substitutions, insertions or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-claudin-6 single domain antibody comprises an amino acid sequence selected from SEQ ID NOs: 7-11 and includes post-translational modifications of the sequence.

[0130] In some embodiments, the single-domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:7, wherein the single-domain antibody binds to claudin-6. In some embodiments, the single-domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:8, wherein the single-domain antibody binds to claudin-6. In some embodiments, the single-domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9, wherein the single-domain antibody binds to claudin-6. In some embodiments, the single-domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10, wherein the single-domain antibody binds to claudin-6.In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:11, wherein the single domain antibody binds to claudin-6.

[0131] For example, functional epitopes can be mapped by combining alanine scanning to identify the amino acids required for interaction with the anti-claudin-6 single domain antibodies herein in the claudin-6 protein. The epitope can be identified using the conformation and crystal structure of the anti-claudin-6 single domain antibody that binds to claudin-6. The present disclosure can provide antibodies that specifically bind to the same epitope as any anti-claudin-6 single domain antibody herein. For example, antibodies are provided that bind to the same epitope as the anti-claudin-6 single domain antibody comprising the amino acid sequence of SEQ ID NO:7. Antibodies are provided that can bind to the same epitope as the anti-claudin-6 single domain antibody comprising the amino acid sequence of SEQ ID NO:8. Antibodies are provided that can bind to the same epitope as the anti-claudin-6 single domain antibody comprising the amino acid sequence of SEQ ID NO:9. Antibodies are provided that can bind to the same epitope as the anti-claudin-6 single domain antibody comprising the amino acid sequence of SEQ ID NO:10. Antibodies are provided that can bind to the same epitope as the anti-claudin-6 single domain antibody comprising the amino acid sequence of SEQ ID NO:11.

[0132] In some embodiments, the present specification provides an anti-claudin-6 antibody or an antigen-binding fragment thereof that specifically binds to claudin-6 and competes with any one of the anti-claudin-6 single-domain antibodies described herein. Competitive binding can be determined by an ELISA assay. For example, an antibody can be provided that specifically binds to claudin-6 and competes with the anti-claudin-6 single-domain antibody comprising the amino acid sequence of SEQ ID NO:7. An antibody can be provided that specifically binds to claudin-6 and competes with the anti-claudin-6 single-domain antibody comprising the amino acid sequence of SEQ ID NO:8. An antibody can be provided that specifically binds to claudin-6 and competes with the anti-claudin-6 single-domain antibody comprising the amino acid sequence of SEQ ID NO:9. An antibody can be provided that specifically binds to claudin-6 and competes with the anti-claudin-6 single-domain antibody comprising the amino acid sequence of SEQ ID NO:10. An antibody can be provided that specifically binds to claudin-6 and competes with the anti-claudin-6 single-domain antibody comprising the amino acid sequence of SEQ ID NO:11.

[0133] In some embodiments, the present specification provides a claudin-6 binding protein comprising any one of the above anti-claudin-6 single domain antibodies. In some embodiments, the claudin-6 binding protein is a monoclonal antibody, including camelid chimeric, humanized or human antibodies. In some embodiments, the anti-claudin-6 antibody is an antibody fragment, for example, a VHH fragment. In some embodiments, the anti-claudin-6 antibody is a full-length antibody consisting only of a heavy chain comprising an Fc region of any antibody class or isotype (e.g., IgG1 or IgG4). The Fc region may have reduced or minimized effector function. In some embodiments, the claudin-6 binding protein is a fusion protein comprising an anti-claudin-6 single domain antibody according to the present specification. The claudin-6 binding protein may be a multispecific antibody comprising an anti-claudin-6 single domain antibody according to the present specification. Other exemplary claudin-6 binding molecules are described in more detail in the following sections.

[0134] In some embodiments, any anti-claudin-6 antibody (e.g., an anti-claudin-6 single domain antibody) or antigen-binding protein according to any of the above embodiments may, alone or in combination, include any feature as described in Sections 5.2.2 to 5.2.7 below.

[0135] 5.2.2. Humanized single domain antibodies The single domain antibodies described herein include humanized single domain antibodies. General strategies for humanizing single domain antibodies from camelid species have been described (see, e.g., Vincke et al., J. Biol. Chem., 284(5):3273-3284 (2009)), and the humanized VHH domains disclosed herein can be used for production. The design of humanized single domain antibodies from camelid species may include signature residues in the VHH, such as residues 11, 37, 44, 45, and 47 (residues numbered according to Kabat) (Muyldermans, Reviews Mol Biotech 74:277-302 (2001)).

[0136] Humanized antibodies, for example, the humanized single domain antibodies disclosed herein, can be produced using various techniques known in the art, including CDR grafting (European Patent No. EP 239,400, International Publication No. WO 91 / 09967, and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596, Padlan, Molecular Immunology 28(4 / 5):489-498(1991), Studnicka et al., Protein Engineering 7(6):805-814(1994), and Roguska et al., PNAS 91:969-973(1994)), chain shuffling (U.S. Patent No. 5,565,332), and techniques disclosed in, for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119-1125(2002), Caldas et al., Protein Eng. 13(5):353-360(2000), Morea et al., Methods 20(3):267-279(2000), Baca et al., J. Biol. Chem. 272(16):10678-10684(1997), Roguska et al., Protein Eng. 9(10):895-904(1996), Couto et al., Cancer Res. 55(23 Supp):5973s-5977s(1995), Couto et al., Cancer Res. 55(8):1717-1722(1995), Sandhu JS, Gene 150(2):409-410(1994), and Pedersen et al. J. Mol. Biol. 235(3):959-973(1994), but not limited thereto. Further, reference is made to U.S. Patent Application No. US 2005 / 004,2664 A1 (February 24, 2005), each of which is hereby incorporated by reference in its entirety.

[0137] In some embodiments, the single domain antibodies according to this specification may be humanized single domain antibodies that bind to claudin-6 (including human claudin-6). For example, the humanized single chain antibodies of the present disclosure may include one or more CDRs shown in SEQ ID NOs: 7-11. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are usually referred to as "import" residues, and these residues are typically taken from the "import" variable domain. For example, humanization can be performed by replacing the corresponding sequences of human antibodies with hypervariable region sequences according to the methods of Jones et al., Nature 321:522-25 (1986), Riechmann et al., Nature 332:323-27 (1988), and Verhoeyen et al., Science 239:1534-36 (1988). As described in Section 6 below, humanization of the single domain antibodies according to this specification is performed.

[0138] In some cases, humanized antibodies are constructed by CDR grafting, where the amino acid sequences of the CDRs of the parental non-human antibody are grafted into a human antibody framework. For example, Padlan et al. have shown that only about one-third of the residues in the CDRs actually contact the antigen, and these are called "specificity-determining residues" or SDRs (Padlan et al., FASEB J. 9:133-39 (1995)). In SDR grafting technology, only the SDR residues are grafted into the human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34 (2005)).

[0139] To reduce antigenicity, the selection of human variable domains for the production of humanized antibodies can be important. For example, by the so-called "best-fit" method, the sequences of non-antibody variable domains are screened against an entire library of known human variable domain sequences. The human sequence that is closest to the non-human antibody may be selected as the human framework of the humanized antibody (Sims et al., J. Immunol. 151:2296-308 (1993) and Chothia et al., J. Mol. Biol. 196:901-17 (1987)). In another method, a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of the light or heavy chain is used. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992) and Presta et al., J. Immunol. 151:2623-32 (1993)). In some cases, the framework is the most abundant human subclass V L 6 subgroup I (V L 6I) and V H subgroup III (V H III) derived from the consensus sequence. In another method, human germline genes are used as a source of framework regions.

[0140] In an alternative paradigm based on the comparison of CDRs, called hyperhumanization, the homology of the FRs is irrelevant. The method involves comparing the non-human sequences to the functional human germline repertoire. Then, genes encoding canonical structures that are the same or closely related to the mouse sequence are selected. Subsequently, within the genes sharing the canonical structure with the non-human antibody, the gene with the highest homology within the CDR is selected as the FR donor. Finally, the non-human CDRs are transplanted into these FRs (see, for example, Tan et al., J. Immunol. 169:1119-25 (2002)).

[0141] Generally, it is desirable to humanize an antibody to retain its affinity for an antigen and other favorably acting biological properties. To achieve this goal, according to one method, a humanized antibody is produced by analyzing the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. A computer program can be obtained that describes and displays the three-dimensional conformation structure of a selected candidate immunoglobulin sequence. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-24 (2002)), Modeller (Sali and Blundell, J. Mol. Biol. 234:779-815 (1993)), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-23 (1997)). By examining these displays, it is possible to analyze the possible role of residues in the function of the candidate immunoglobulin sequence, for example, the analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. Thus, by selecting and combining FR residues from the receptor and import sequences, necessary antibody characteristics such as an increase in affinity for one or more target antigens can be obtained. Usually, in terms of affecting antigen binding, the hypervariable region residues are directly and most prominently involved.

[0142] Another method of antibody humanization is based on a measure of the degree of antibody humanization called Human String Content (HSC). This method compares the mouse sequence to the human germline gene repertoire and scores the differences as HSC. Then, instead of using a global identity measurement, the target sequence is humanized by maximizing its HSC to produce multiple different humanized variants (Lazar et al., Mol. Immunol. 44:1986-98 (2007)).

[0143] In addition to the above methods, empirical methods can also be used to produce and select humanized antibodies. These methods include those based on the generation of large libraries of humanized variants and the selection of optimal clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, for example, Hoogenboom, Nat. Biotechnol. 23:1105-16 (2005), Dufner et al., Trends Biotechnol. 24:523-29 (2006), Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003), and Schlapschy et al., Protein Eng. Des. Sel. 17:847-60 (2004)).

[0144] In the FR library method, a block of residue variants is introduced at specific positions within the FR, and the FR that best supports the transplanted CDR is selected by screening the library. The residues to be substituted may include some or all of the "Bernea" residues identified as potentially contributing to the CDR structure (see, for example, Foote and Winter, J. Mol. Biol. 224:487-99 (1992)), or a more limited group of target residues from the identification of Baca et al., J. Biol. Chem. 272:10678-84 (1997).

[0145] In FR shuffling, the entire FR is combined with non-human CDRs without creating a combinatorial library of selected residue variants (see, for example, Dall’Acqua et al., Methods 36:43-60 (2005)). A one-step FR shuffling process may be used. Such methods have proven to be effective because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, for example, Damschroder et al., Mol. Immunol. 44:3049-60 (2007)).

[0146] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and on the evaluation of the sequential replacement and ligation of non-human fragments into a human FR library. This method typically causes the epitope to retain and identify antibodies from multiple subclasses having different human V segment CDRs.

[0147] The "human engineering" method relates to producing a modified antibody having reduced immunogenicity in humans by making specificity modifications to the amino acid sequence of an antibody to modify a non-human antibody or antibody fragment, the modified antibody still retaining the required binding properties of the original non-human antibody. Typically, the technique relates to classifying amino acid residues of a non-human antibody as "low risk", "intermediate risk" or "high risk" residues. The classification is done using an overall risk / return calculation that evaluates the predicted benefit of a particular substitution (e.g., to human immunogenicity) and the risk that the substitution will affect the folding of the resulting antibody. By aligning the amino acid sequence from the non-human antibody variable region with the corresponding region of a specific or consensus human antibody sequence, specific human amino acid residues that are to be substituted at a given position (e.g., low risk or intermediate risk) in the non-human antibody sequence can be selected. Amino acid residues at low risk or intermediate risk positions in the non-human sequence can be substituted with the corresponding residues in the human antibody sequence by alignment. Techniques for making human engineered proteins are described in Studnicka et al., Protein Engineering 7:805-14 (1994), U.S. Patents Nos. 5,766,886, 5,770,196, 5,821,123 and 5,869,619, and PCT Publication WO 93 / 11794.

[0148] Composite human antibodies can be produced, for example, using the Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To produce a composite human antibody, the immunogenicity of the resulting antibody is minimized by designing variable region sequences from fragments of multiple human antibody variable region sequences so as to avoid T cell epitopes.

[0149] An immunodeleted antibody is an antibody in which the T cell epitopes therein have been removed. Methods for manufacturing immunodeleted antibodies are described. See, for example, Jones et al., Methods Mol Biol. 525:405-23 (2009), xiv and De Groot et al., Cell. Immunol. 244:148-153 (2006). An immunodeleted antibody contains a variable region lacking T cell epitopes and a human constant region. Briefly, the variable region of an antibody is cloned and then T cell epitopes are identified by testing overlapping peptides derived from the antibody variable region in a T cell proliferation assay. T cell epitopes are identified by computer methods to identify peptides that bind to human MHC class II. Mutations are introduced into the variable region to remove binding to human MHC class II. Then, an immunodeleted antibody is produced using the mutated variable region.

[0150] 5.2.3. Single domain antibody variants In some embodiments, one or more amino acid sequence modifications of the single-domain antibodies that bind to Claudin-6 described herein were considered. For example, optimization of the binding affinity and / or other biological properties of the antibody may be required, including, but not limited to, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Therefore, in addition to the single-domain antibodies that bind to Claudin-6 described herein, it is also expected to produce variants of the single-domain antibodies that bind to Claudin-6 described herein. For example, single-domain antibody variants can be produced by introducing appropriate nucleotide changes into the coding DNA and / or synthesizing the desired antibody or polypeptide. Those skilled in the art will understand that amino acid modifications can alter the post-translational processes of single-domain antibodies.

[0151] Chemical modification In some embodiments, the single-domain antibodies according to the present specification are chemically modified, for example, covalently bound to the single-domain antibody via any type of molecule. Antibody derivatives may include, for example, antibodies that have already been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic hydrolysis and cleavage, ligation with cell ligands or other proteins, or conjugation with one or more immunoglobulin domains (e.g., Fc or a portion of Fc). Any one of the plurality of chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Also, the antibody may contain one or more non-classical amino acids.

[0152] The antibodies according to the present specification can be modified to increase or decrease the degree of glycosylation of the antibody. By modifying the amino acid sequence to produce or remove one or more glycosylation sites, addition or deletion of antibody glycosylation sites can be easily achieved.

[0153] When the single-domain antibody according to this specification is fused with the Fc region, the carbohydrate linked thereto may be modified. Natural antibodies produced from mammalian cells usually contain branched biantennary oligosaccharides, which are usually linked to Asn297 of the CH2 domain of the Fc region via an N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may contain various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose linked to GlcNAc in the "stem" of the biantennary oligosaccharide structure. Modifications can be made to the oligosaccharide in the binding molecule according to this specification to produce variants with several improved properties.

[0154] In other embodiments, when the single domain antibodies according to the present specification are fused to an Fc region, the antibody variants according to the present specification may have a carbohydrate structure lacking fucose (either directly or indirectly) linked to the Fc region. For example, the amount of fucose in such an antibody may be 1% - 80%, 1% - 65%, 5% - 65% or 20% - 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at position Asn297 relative to the total of all sugar structures (e.g., complex, hybrid and high mannose structures) linked to Asn297, measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU number of Fc region residues) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 and US 2004 / 0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, US 2003 / 0115614, US 2002 / 0164328, US 2004 / 0093621, US 2004 / 0132140, US 2004 / 0110704, US 2004 / 0110282, US 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing afucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986), US Patent Application No. US 2003 / 0157108, and WO 2004 / 056312, particularly Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO 2003 / 085107).

[0155] Binding molecules comprising single domain antibodies according to the present specification are further provided with oligosaccharides to be divided, for example, here, the bi-contact angle oligosaccharide connected to the Fc region is divided by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), US Patent No. 6,602,684 (Umana et al.) and US 2005 / 0123546 (Umana et al.). Further provided are variants having at least one galactose residue in the oligosaccharide linked to the Fc region. Such variants may have improved CDC function. Such variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0156] In the molecule comprising the single domain antibody and the Fc region of the present invention, Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region. The Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0157] In the present application, variants having some (but not all) effector functions are contemplated, which functions render the antibody variants desirable candidates for applications in which the in vivo half-life of the binding molecule is important, but in which some effector functions (e.g., complement and ADCC) are not necessary or are detrimental. Reduction / depletion of CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, by performing an Fc receptor (FcR) binding assay, it can be ensured that the binding molecule does not have FcγR binding ability (and thus may lack ADCC activity), but can retain FcRn binding ability. See U.S. Patent No. 5,500,362 (e.g., see Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985), 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)) for non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule. Alternatively, non-radioactive assay methods may be used, e.g., see the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, in vivo, the ADCC activity of the target molecule may be evaluated in an animal model, e.g., as disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). By performing a C1q binding assay, it can be confirmed that the antibody cannot bind to C1q and thus lacks CDC activity.See, for example, the ELISA for C1q and C3c binding in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination may be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0158] Binding molecules with reduced effector function include antibodies (U.S. Patent No. 6,737,056) with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329. Such Fc mutants include Fc mutants with substitutions of two or more at amino acid positions 265, 269, 270, 297, and 327, including the Fc mutant designated "DANA" in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0159] Several mutants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0160] The variant may comprise an Fc region having one or more amino acid substitutions, and these substitutions (e.g., substitutions at positions 298, 333 and / or 334 within the Fc region (EU numbers of the residues)) improve ADCC. Modifications may occur within the Fc region, thereby causing a modification (i.e., improvement or decrease) in C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent No. 6,194,551, WO 99 / 51642 and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0161] Binding molecules having an extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These molecules comprise an Fc region having one or more amino acid substitutions, where these substitutions improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more Fc region residues of 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, those that substitute Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 relating to other examples of Fc region variants.

[0162] Production of cysteine engineered antibodies may be required, where one or more residues of the antibody are replaced by cysteine residues. The replaced residues may be present at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein.

[0163] Substitution, deletion or insertion The mutations may be substitutions, deletions or insertions of one or more codons encoding a single domain antibody or polypeptide, which cause alterations in the amino acid sequence relative to the original antibody or polypeptide. The target sites for substitution mutagenesis include CDRs and FRs.

[0164] The amino acid substitutions may be the result of replacing an amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, for example, a conservative amino acid substitution. Standard techniques well known to those skilled in the art may be used to introduce mutations into the nucleotide sequences encoding the molecules herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that cause amino acid substitutions. The insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The substitutions, deletions or insertions may include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to the original molecule. The substitutions may be conservative amino acid substitutions at one or more predicted non-essential amino acid residues. The allowable changes can be determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the activity exhibited by the parental antibody of the resulting variant.

[0165] Insertion of an amino acid sequence includes fusion of the amino and / or carboxyl termini within the scope of a polypeptide containing from one residue to multiple residues in length, and insertion within the sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue.

[0166] The present disclosure includes single domain antibodies generated by conservative amino acid substitutions. In conservative amino acid substitutions, an amino acid residue is substituted with an amino acid residue having a side chain with a similar charge. As noted above, in the art, families of amino acid residues having side chains with similar charges are defined. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations may be introduced randomly along all or part of the coding sequence, e.g., by saturation mutagenesis, and the biological activity of the resulting mutants screened to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein determined. Conservative (e.g., within groups of amino acids having similar properties and / or side chains) substitutions may be made to retain or not significantly alter the properties. Exemplary substitutions are shown in Table 2 below.

[0167] [Table 2]

[0168] Amino acids can be grouped based on the similarity of the properties of their side chains (see, for example, Lehninger, Biochemistry 73-75 (2nd ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), (3) acidic: Asp (D), Glu (E), and (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) acidic: Asp, Glu, (4) basic: His, Lys, Arg, (5) residues affecting chain orientation: Gly, Pro, and (6) aromatic: Trp, Tyr, Phe. For example, any cysteine residue not involved in maintaining the correct conformation of a single domain antibody may be substituted with another amino acid, such as alanine or serine, in order to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Non-conservative substitutions would require the exchange of one member in these classes for another.

[0169] One type of substitution variant relates to the substitution of one or more residues in the hypervariable regions of a parent antibody (e.g., a humanized or human antibody). Typically, one or more of the resulting variants are selected for further study, and modified (e.g., improved) with respect to the parent antibody in terms of some biological properties (e.g., increased affinity, decreased immunogenicity), and / or substantially retain some biological properties of the parent antibody. Exemplary substitution variants are, for example, affinity matured antibodies that can be readily generated by affinity maturation techniques based on phage display (e.g., those techniques described herein). Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0170] Modifications (e.g., substitutions) can be made in the CDRs to, for example, improve antibody affinity. Such modifications may be made in CDR “hot spots,” i.e., residues encoded by codons that mutate frequently during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in SDR (a-CDR), where the binding affinity of the resulting mutant antibody or fragment thereof is tested. Affinity maturation by construction and reselection from a secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O’Brien et al., Human Press, Totowa, NJ, (2001)). In some cases of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide site-directed mutagenesis). A secondary library is then created. The library is screened to identify any antibody variants having the desired affinity. Another method of introducing diversity relates to the CDR-guided method, where some CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, by alanine scanning mutagenesis or modeling. More detailed descriptions regarding affinity maturation are provided in the following sections.

[0171] Substitutions, insertions, or deletions may occur within one or more CDRs, provided that such modifications do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative modifications (e.g., conservative substitutions as defined herein) that do not substantially reduce binding affinity may be made in the CDRs. In some embodiments of the mutant VHH sequences as defined herein, each CDR is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0172] As described in Cunningham and Wells, Science, 244:1081-1085 (1989), a useful method for identifying residues or regions of an antibody capable of performing targeted mutagenesis is called "alanine scanning mutagenesis." In this method, residues or groups of residues of a target residue (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified, and substitution is performed using a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. By introducing another substitution at the position of the amino acid, the functional sensitivity to the initial substitution can be demonstrated. Alternatively, or additionally, the crystal structure of the antigen-antibody complex can be determined to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted as substitution candidates or removed. The variants may be screened to determine whether they contain the desired properties.

[0173] Insertions of amino acid sequences include amino-terminal and / or carboxy-terminal fusions within the range of polypeptides having lengths from one residue to one hundred or more residues, and insertions of sequences of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion mutants of the antibody molecule include fusing the N-terminus or C-terminus of the antibody with an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of the antigen-binding domain antibody.

[0174] The changes may be made using known methods in the art such as oligonucleotide-mediated (site-specific) mutagenesis, alanine scanning, and PCR mutagenesis. To produce single-domain antibody variants DNA, site-specific mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986) and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)) or other known techniques may be performed on the cloned DNA.

[0175] 5.2.4. In Vitro Affinity Maturation Antibody variants with improved properties such as affinity, stability, or expression level compared to the parental antibody can be produced by in vitro affinity maturation. Similar to the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Antibody libraries are displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cells) or associated (e.g., covalently or non-covalently) with their encoded mRNA or DNA. Selection of the affinity of the displayed antibody allows for the isolation of the organism or complex having the genetic information encoding the antibody. Two or three rounds of mutation and selection using a display method such as phage display typically produce antibody fragments with affinities in the low nanomolar range. Affinity matured antibodies can have nanomolar or picomolar affinities for the target antigen.

[0176] Phage display is a common method for displaying and selecting antibodies. Antibodies are displayed on the surface of Fd or M13 phage as fusions with phage coat proteins. Selection is with respect to the binding of the antibodies to their targets that are exposed to the antigen, and the process is called "panning". Phage that bind to the antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002) and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0177] In the yeast display system (see, for example, Boder et al., Nat. Biotech. 15:553-57 (1997) and Chao et al., Nat. Protocols 1:755-68 (2006)), an antibody can be fused to the adhesion subunit of the yeast lectin protein Aga2p, and the adhesion subunit is linked to the yeast cell wall via a disulfide bond with Aga1p. The Aga2p display protein minimizes potential interactions with other molecules on the yeast cell wall by protruding the protein from the cell surface. Magnetic separation and flow cytometry are used to screen libraries to select antibodies with improved affinity or stability. Yeast is labeled with a biotinylated antigen and a second reagent conjugated to a fluorophore, such as streptavidin, to measure binding to the target soluble antigen. The change in the expression on the antibody surface is measured by immunofluorescent labeling of hemagglutinin or c-Myc epitope tags (e.g., scFv) located on both sides of the single-chain antibody. Expression has been shown to be related to the stability of the displayed protein, and thus antibodies can be selected to improve stability and affinity (see, for example, Shusta et al., J. Mol. Biol. 292:949-56 (1999)). Another advantage of yeast display is that it utilizes the endoplasmic reticulum chaperone and quality control mechanisms, and the displayed protein is folded in the endoplasmic reticulum of eukaryotic yeast cells. Once maturation is complete, the antibody affinity can be easily "titrated" and displayed on the yeast surface, eliminating the need to express and purify each clone. The theoretical limitation of yeast surface display is the potentially smaller functional display size than other display methods. However, recent methods use the mating system of yeast cells to generate combinatorial diversity of about 10 14 which is (see, for example, US Patent Publication 2003 / 0186374 and Blaise et al., Gene 342:211-18 (2004)).

[0178] In ribosome display, antibody-ribosome-mRNA (ARM) complexes are produced and used for selection in a cell-free system. A DNA library encoding a specific antibody library is fused with a spacer sequence gene lacking a stop codon. The spacer sequence remains ligated to the peptide tRNA during translation and occupies the ribosome channel, protruding and folding the target protein from the ribosome. The resulting complex of mRNA, ribosome, and protein can capture and isolate the antibody and its encoding mRNA by binding to a surface-bound ligand through affinity for the ligand. Then, the mRNA bound to the ribosome can be reverse transcribed into cDNA and subjected to mutagenesis for use in the next round of selection (see, for example, Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, puromycin is used as an adapter molecule to establish a covalent bond between the antibody and the mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0179] Since these methods are performed entirely in vitro, they offer two main advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, since there is no need to transform the library after any diversification step, random mutations can be easily introduced, for example, by a non-proofreading polymerase, after each round of selection.

[0180] A mammalian display system may also be used.

[0181] Diversity can also be introduced into the CDRs of an antibody library by targeted or random introduction. The former method involves targeting all the CDRs of an antibody in sequence by high or low levels of mutagenesis, or targeting isolated hotspots of somatic hypermutation (see, e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)), or suspecting residues that affect affinity based on experimental or structural reasons. Diversity may also be introduced by replacing natural diversification regions by DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003), U.S. Pat. Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops that extend to framework region residues (see, e.g., Bond et al., J. Mol. Biol. 348:699-709 (2005)), use loop deletions and insertions in the CDRs, or use hybridization-based diversification (see, e.g., U.S. Patent Publication No. 2004 / 0005709). Other methods of generating diversity in the CDRs are disclosed, for example, in U.S. Pat. No. 7,985,840. Other methods that can be used for generating antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Pat. Nos. 8,685,897 and 8,603,930 and U.S. Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855 and 2009 / 0075378, which are hereby incorporated by reference in their entirety.

[0182] Screening of libraries can be accomplished by a variety of techniques known in the art. For example, single domain antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed in host cells attached to adsorption plates or used for cell sorting, or conjugated to biotin and captured with streptavidin-coated beads, or used in any other method to pan display libraries.

[0183] For an overview of methods of in vitro affinity maturation, see, for example, Hoogenboom, Nature Biotechnology 23:1105-16 (2005), Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010), and references therein.

[0184] 5.2.5. Modification of single domain antibodies Covalent modification of single domain antibodies is included within the scope of the present disclosure. Covalent modification includes reacting a targeted amino acid residue of the single domain antibody with an organic derivatizing agent capable of reacting with a predetermined side chain or N-terminal or C-terminal residue of the single domain antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to their corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, for example, Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0185] Other types of covalent modifications of the single-domain antibodies included within the scope of the present disclosure include altering the native glycosylation pattern of the above-described antibodies or polypeptides (e.g., Beck et al., Curr. Pharm. Biotechnol. 9:482-501 (2008), and Walsh, Drug Discov. Today 15:773-80 (2010)), e.g., conjugating the antibody to one of a plurality of non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene, by the methods described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. The single-domain antibodies that bind to claudin-6 of the present disclosure can further be fused or conjugated with one or more immunoglobulin constant regions or portions thereof (e.g., Fc) genes to extend the half-life and / or confer known Fc-mediated effector functions.

[0186] Also, single-chain antibodies that bind to claudin-6 of the present disclosure can be modified to form chimeric molecules, and these chimeric molecules include single-chain antibodies that bind to claudin-6 and are fused to another heterologous polypeptide or amino acid sequence, such as an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (edited by Chamow and Ashkenazi, 1999)). The single-chain antibodies that bind to claudin-6 may be used to generate chimeric antigen receptors (CARs) that bind to claudin-6, as will be described in more detail below.

[0187] The present specification further provides a fusion protein, which comprises a single-chain antibody that binds to the claudin-6 of the present disclosure and a heterologous polypeptide. In some embodiments, the heterologous polypeptide that is fused or chemically conjugated to the antibody gene can be used to target the antibody to cells having claudin-6 expressed on the cell surface.

[0188] The present specification further provides a set of antibodies that bind to the claudin-6 antigen. The set of antibodies can have different binding rates, different dissociation rates, different affinities for the claudin-6 antigen, and / or different specificities for the claudin-6 antigen. The set can comprise or consist of about 10 to about 1000 or more antibodies. The set of antibodies can be used, for example, in a 96-well or 384-well plate for measurements such as ELISA.

[0189] 5.2.6. Production of single-domain antibodies Methods for preparing single-domain antibodies are described. See, for example, Els Pardon et al., Nature Protocol, 9(3): 674 (2014). Single-domain antibodies (e.g., VHH) can be obtained by methods known in the art, for example, immunizing camelid species (e.g., camels or llamas), then obtaining hybridomas, or cloning a single-domain antibody library by molecular biology techniques known in the art, and then selecting by ELISA using a single clone or phage display of the unselected library.

[0190] The single-domain antibodies according to the present specification can be produced by culturing cells transformed or transfected with a vector containing a nucleic acid encoding the single-domain antibody. The polynucleotide sequence encoding the polypeptide component of the antibody of the present disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells such as hybridoma cells or B cells. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a host cell. Many known vectors available in the art can be used for the purposes of the present disclosure. The selection of an appropriate vector depends mainly on the size of the nucleic acid inserted into the vector and the particular host cell to be transformed with the vector. Host cells suitable for expressing the antibodies of the present disclosure include prokaryotes such as archaebacteria and eubacteria including gram-negative or gram-positive organisms, eukaryotic microorganisms such as filamentous fungi or yeasts, invertebrate cells such as insect or plant cells, and vertebrate cells such as mammalian host cell lines. The host cells are transformed with the above expression vector and cultured in a conventional nutrient medium, which is appropriately modified to induce the promoter, select the transformant, or amplify the gene encoding the necessary sequence. The antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0191] Antibody production methods, including vector construction, expression, and purification, are further described in the literature of Pluckthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (edited by McCafferty et al., 1996), Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, Current Protocols in Protein Science (2009), Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, Antibody Expression and Production (edited by Al-Rubeai, 2011), and Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009).

[0192] Of course, it is expected that alternative methods well known in the art can be used to produce anti-claudin-6 single-domain antibodies. For example, an appropriate amino acid sequence or a part thereof can be produced by direct peptide synthesis using solid-phase techniques (see, for example, Stewart et al., Solid-Phase Peptide Synthesis (1969) and Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963)). Protein synthesis in vitro can be performed using manual techniques or by automation. Each part of the anti-claudin-6 antibody can be chemically synthesized individually and combined by chemical or enzymatic methods to produce the required anti-claudin-6 antibody. Alternatively, the antibody can be purified from the cells or body fluids, such as milk, of transgenic animals expressing the engineered antibody, as disclosed in U.S. Pat. Nos. 5,545,807 and 5,827,690.

[0193] Specifically, the single-domain antibodies or other claudin-6 binding proteins according to this specification can be generated by the following method: immunize llamas, select single B cells, extract V genes, clone claudin-6 binding proteins (such as VHH-Fc fusions), and perform small-scale expression and purification. Additionally, another screening may be performed on single-domain antibodies and other molecules that bind to claudin-6, including selecting one or more of ELISA positive, BLI positive, and K less than 100 nM. These selection criteria can be combined as described in Section 6 below. Also, the binding ability of a single VHH binding protein (and other molecules that bind to claudin-6) to cells expressing claudin-6 can be measured. Such measurements can be performed by analyzing cells expressing claudin-6 using FACS and measuring the mean fluorescence intensity (MFI) of fluorescently labeled VHH molecules. Each of the above aspects will be described in more detail below. D This includes selecting one or more of the above. These selection criteria can be combined as described in Section 6 below. Also, the binding ability of a single VHH binding protein (and other molecules that bind to claudin-6) to cells expressing claudin-6 can be measured. Such measurements can be performed by analyzing cells expressing claudin-6 using FACS and measuring the mean fluorescence intensity (MFI) of fluorescently labeled VHH molecules. Each of the above aspects will be described in more detail below.

[0194] Polyclonal antibody Polyclonal antibodies are usually produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Bifunctional or derivatizing agents, such as maleimidobenzoyl sulfosuccinimide ester (conjugated via cysteine residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR (where R and R 1Independently, a related antigen is conjugated with a protein having immunogenicity in the immunized species, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a lower alkyl group (). Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dimycolate). The immunization scheme can be selected by those skilled in the art without undue experimentation.

[0195] For example, for animals, immunization against an antigen, immunogenic conjugate, or derivative is carried out by combining, for example, 100 μg or 5 μg of protein or conjugate (used for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and intradermally injecting the solution at multiple sites. One month later, a booster immunization is carried out on the animals using 1 / 5 to 1 / 10 of the amount of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, blood is collected from the animals and the antibody titer of the serum is measured. The animals are boosted until the titer plateau. Conjugates can also be produced as protein fusions in recombinant cell cultures. Note that aggregating agents such as alum are suitable for enhancing the immune response.

[0196] Monoclonal antibody Monoclonal antibodies are obtained from a substantially homogeneous group of antibodies, i.e., each antibody constituting the group is the same except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Therefore, the modifier "monoclonal" indicates the characteristic of the antibody that it is not a mixture of individual antibodies.

[0197] For example, monoclonal antibodies may be produced by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0198] In the hybridoma method, an appropriate host animal is immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing protein. Alternatively, the lymphocytes may be immunized in vitro. Then, the lymphocytes are fused with myeloma cells using an appropriate fusing agent such as polyethylene glycol to generate hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103 (Academic Press, 1986)).

[0199] The immunizing agent usually contains the antigen protein or a fusion variant thereof. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pages 59-103. The immortalized cell line is usually a transformed mammalian cell. The hybridoma cells thus produced are inoculated into an appropriate medium and grown therein, and the medium may contain one or more substances that inhibit the growth or survival of unfused parental myeloma cells. Preferred immortalized myeloma cell lines are cells that fuse efficiently, support the stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to media such as HAT medium.

[0200] Measure the production of monoclonal antibodies against the antigen in the medium in which the hybridoma cells grow. The presence of monoclonal antibodies against the desired antigen in the medium in which the hybridoma cells are cultured can be measured. Such techniques and measurements are known in the art. For example, the binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0201] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by the limiting dilution method and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Note that hybridoma cells can grow as tumors in vivo in mammals.

[0202] Monoclonal antibodies secreted by subclones can be appropriately isolated from the medium, ascites, or serum by conventional immunoglobulin purification methods such as protein A-agarose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0203] Monoclonal antibodies can also be produced by recombinant DNA methods as described in U.S. Patent No. 4,816,567 and supra. Using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of a mouse antibody), the DNA encoding the monoclonal antibody can be easily isolated and sequenced. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is inserted into an expression vector and transfected into host cells such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins, so that monoclonal antibodies can be synthesized in such recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

[0204] Antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991). Subsequent publications have described strategies for constructing very large phage libraries that produce high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)) and combinatorial infection and in vivo recombination (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Thus, these techniques are a viable alternative to traditional monoclonal antibody hybridoma technology for isolating monoclonal antibodies.

[0205] DNA can be modified, for example, by substituting the coding sequence (U.S. Patent No. 4,816,567, Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the coding sequence. For producing chimeric bivalent antibodies, such non-immunoglobulin polypeptides may be substituted and the chimeric bivalent antibodies include one antigen-binding site specific for an antigen and a further antigen-binding site specific for a different antigen.

[0206] Chimeric or hybrid antibodies can also be produced in vitro using known methods in synthetic protein chemistry, including those methods regarding cross-linking agents. For example, immunotoxins can be constructed by forming disulfide exchange reactions or thioether bonds. Examples of suitable reagents used for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0207] Recombinant production in prokaryotic cells The polynucleotide sequences encoding the antibodies of the present disclosure can be obtained by standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a prokaryotic host. Many known vectors available in the art can be used for the purposes of the present disclosure. The selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains different components, which depend on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the host cell in which it resides. Vector components typically include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0208] Typically, plasmid vectors containing a replicon and control sequences derived from a species compatible with the host cell are used in combination with these hosts. The vector usually has a replication site and a label sequence that can provide phenotypic selection in the transformed cell. For example, E. coli is typically transformed with pBR322 (a plasmid derived from the E. coli species). Examples of pBR322 derivatives for expressing specific antibodies are described in detail in U.S. Patent No. 5,648,237 to Carter et al.

[0209] Note that phage vectors containing a replicon and control sequences compatible with the host microorganism can be used as transformation vectors associated with these hosts. For example, phages such as GEM (trademark)-11 can be used to produce recombinant vectors, which can be used to transform susceptible host cells such as E. coli LE392.

[0210] The expression vector of the present application may contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is an untranslated regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are usually classified into two types: inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the transcription level of the cistron under its control in response to changes in culture conditions (e.g., the presence or absence of nutrients, or changes in temperature).

[0211] A number of promoters recognized by many potential host cells are well-known. The selected promoter can be functionally linked to the cistron DNA encoding the antibody of the present invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application. Both natural promoter sequences and a plurality of heterologous promoters can be used to guide the amplification and / or expression of the target gene. Heterologous promoters can be used because they usually allow for greater transcription and higher yields of the target gene to be expressed compared to natural target polypeptide promoters.

[0212] Promoters suitable for prokaryotic hosts include the PhoA promoter, the β-galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters acting on bacteria (e.g., other known bacterial or phage promoters) are also suitable. Since their nucleic acid sequences have been disclosed, those skilled in the art can functionally link them to the cistron encoding the target peptide using linkers or adapters (Siebenlist et al. Cell 20: 269 (1980)) to provide any necessary restriction sites.

[0213] In one aspect, each cistron within the recombinant vector comprises a secretory signal sequence component that guides the transmembrane translocation of the expressed polypeptide. Typically, the signal sequence may be a component of the vector or part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present disclosure should be a signal sequence that can be recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence may be replaced with a prokaryotic signal sequence, which is selected from, for example, alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP.

[0214] Since the production of antibodies according to the present disclosure can occur in the cytoplasm of the host cell, it is not necessary to have a secretory signal sequence for each cistron. Some host strains (e.g., E. coli trxB - strains) provide cytoplasmic conditions favorable for the formation of disulfide bonds, enabling the correct folding and assembly of the expressed protein subunits.

[0215] Prokaryotic host cells suitable for expressing the antibodies of the present disclosure include archaebacteria and eubacteria such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla, or Paracoccus. In some embodiments, Gram-negative cells are used. E. coli cells can be used as a host. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Volume 2 (Washington, D.C.: American Society for Microbiology, 1987), pages 1190-1219, ATCC Deposit No. 27,325) and its derivatives, genotype W3110 ΔfhuA (ΔtonA) ptr3 lacIq lacL8 ΔompT Δ(nmpc-fepE) degP41 kan R including strain 33D3 having (U.S. Patent No. 5,639,635). Other strains and their derivatives, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608) are also suitable. These examples are for illustrative purposes only and are not limiting. Methods for constructing derivatives of any of the above bacteria having a defined genotype are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). Usually, it is necessary to select a suitable bacterium considering the replicability of the replicon in the bacterial cell. For example, when supplying a replicon with a well-known plasmid (e.g., pBR322, pBR325, pACYC177, or pKN410), E. coli, Serratia, or Serratia species can be preferably used as a host.

[0216] Typically, the host cell should secrete a minimal amount of proteolytic enzymes and should be able to ideally incorporate another protease inhibitor into the cell culture.

[0217] The host cell is transformed with the above expression vector and cultured in a conventional nutrient medium, which is appropriately modified to induce the promoter, select the transformant, or amplify the gene encoding the required sequence. Transformation refers to introducing DNA into a prokaryotic host such that the DNA can replicate as an extrachromosomal element or via chromosomal integration. Depending on the host cell used, transformation is carried out using standard techniques suitable for these cells. Calcium treatment using calcium chloride is usually used for bacterial cells containing a large amount of cell wall barriers. Another transformation method is using polyethylene glycol / DMSO. Another technique used is electroporation.

[0218] Prokaryotic cells suitable for producing the antibodies of the present application grow in media known in the art and are suitable for culturing the selected host cells. Examples of suitable media include luria broth (LB) and the necessary nutrient supplements. The medium may further contain a selection agent selected based on the construction of the expression vector, thereby selectively allowing the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells expressing the ampicillin resistance gene.

[0219] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may be introduced at appropriate concentrations, either alone or as a mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the medium may contain one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycolate, dithiothreitol, and dithioseitol. The prokaryotic host cells are cultured at an appropriate temperature and pH.

[0220] When an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one aspect of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, the transformed host cells are cultured and induced in a phosphate-limited medium. For example, the phosphate-limited medium is a C.R.A.P medium (see, for example, Simmons et al., J. Immunol. Methods 263:133-147 (2002)). Depending on the vector construct used, a plurality of other inducers known in the art can be used.

[0221] The antibodies expressed in the present disclosure are secreted into the periplasm of the host cell and then recovered. Protein recovery usually involves the disruption of microorganisms and usually uses methods such as osmotic shock, sonication or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the medium and then isolated. The cells can be removed from the medium, filtered, and the culture supernatant concentrated to further purify the produced protein. The expressed polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0222] Instead, proteins are produced in large quantities by fermentation methods. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. To increase the yield and quality of the antibodies of the present disclosure, various fermentation conditions can be varied. For example, chaperones have been shown to assist in the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. J Bio Chem 274:19601-19605 (1999), U.S. Patent No. 6,083,715, U.S. Patent No. 6,027,888, Bothmann and Pluckthun, J. Biol. Chem. 275:17100-17105 (2000), Ramm and Pluckthun, J. Biol. Chem. 275:17106-17113 (2000), Arie et al., Mol. Microbiol. 39:199-210 (2001).

[0223] To minimize proteolysis of the heterologous protein being expressed (especially those sensitive to proteolysis), several host strains lacking proteolytic enzymes can be used in the present disclosure, for example, as described in U.S. Patent No. 5,264,365, U.S. Patent No. 5,508,192, Hara et al., Microbial Drug Resistance, 2:63-72 (1996). An E. coli strain transformed with a plasmid overexpressing one or more chaperones and lacking proteolytic enzymes can be used as a host cell in the expression system encoding the antibody of the present application.

[0224] The antibodies produced in this specification can be further purified to obtain a substantially homogeneous preparation and used for further measurement and use. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse phase HPLC, silica or cation exchange resin chromatography, such as chromatography on DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75. Protein A immobilized on a solid phase can be used, for example, in some embodiments, for the immunoaffinity purification of the binding molecules of the present disclosure. The solid phase on which protein A is immobilized may be a column containing a glass or silica surface, or a controlled microporous glass or silicate column. The column may be coated with a reagent such as glycerol to prevent non-specific attachment of contaminants. Then the solid phase is washed to remove contaminants that bind non-specifically to the solid phase. Finally, the desired antibody is recovered from the solid phase by elution.

[0225] Recombinant production in eukaryotic cells For eukaryotic expression, vector components typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more selectable marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0226] Vectors used in eukaryotic hosts may be inserts of a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected may be a heterologous signal sequence that is recognized and processed by the host cell (i.e., cleaved by signal peptidase). In mammalian cell expression, mammalian signal sequences and viral secretion leader sequences, such as the herpes simplex gD signal, can be obtained. DNA of such a precursor region can be ligated to the DNA encoding the antibody of the present application in the reading frame.

[0227] Typically, mammalian expression vectors do not require an origin of replication component (the SV40 origin contains an early promoter and is usually not usable).

[0228] Expression and cloning vectors may contain a selectable gene, also called a selectable marker. The selectable gene encodes a protein that confers resistance to an antibiotic or other toxin (e.g., ampicillin, neomycin, methotrexate or tetracycline), complements an auxotrophic defect, or provides an essential nutrient not obtainable from complex media.

[0229] In one example of the selection form, a drug blocks the growth of host cells. Cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive in the selection form. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0230] Another example of a suitable selectable marker for use in mammalian cells is one that can identify cells that can take up the nucleic acid encoding the antibody of the present application. For example, cells transformed with a DHFR selectable gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When using wild-type DHFR, an exemplary suitable host cell line is a Chinese hamster ovary (CHO) cell line lacking DHFR activity. Alternatively, host cells transformed or co-transformed with a polypeptide encoding a DNA sequence, a wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) (especially wild-type hosts containing endogenous DHFR) can be selected by cell growth in a medium containing a selection agent used for the selectable marker, e.g., an aminoglycoside antibiotic.

[0231] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding the required polypeptide sequence. Eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream of the transcription start site. It may also contain another sequence found 70-80 bases upstream of the transcription start of many genes. The 3' end of most eukaryotes may be a signal for adding a polyadenylic acid tail to the 3' end of the coding sequence. Any of these sequences can be inserted into a eukaryotic expression vector.

[0232] Transcription of the vector's polypeptide from mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses (such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40)), heterologous mammalian promoters (such as the actin promoter or immunoglobulin promoter), and heat shock promoters, provided that these promoters are compatible with the host cell line.

[0233] Transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotes is usually increased by inserting enhancer sequences into the vector. Currently, many enhancer sequences from mammalian genes (such as globin, elastase, albumin, alpha-fetoprotein, and insulin) are known. Examples include the SV40 enhancer (bp 100-270) located on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located on the late side of the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297:17-18 (1982) for enhancer elements for activating eukaryotic promoters. Enhancers can be spliced into the vector at the 5' or 3' position of the polypeptide coding sequence, but are preferably located at the 5' site of the promoter.

[0234] Expression vectors for use in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences can usually be obtained from the 5', and in some cases the 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide fragments that are transcribed into polyadenylated fragments in the untranslated portion of the polypeptide encoded by the mRNA. A useful transcription termination component is the bovine growth hormone polyadenylation region.

[0235] Suitable host cells for cloning or expressing DNA in the vectors of this specification include the higher eukaryotic cells described herein, including vertebrate host cells. In culture (tissue culture), the growth of vertebrate cells follows common procedures. Examples of useful mammalian host cell lines include the monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651), the human fetal kidney cell line (293 or 293 cell subclones are used for growth in suspension medium, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells, and the human liver cancer cell line (Hep G2).

[0236] The host cell can be transformed with the above expression or cloning vector for producing an antibody, cultured in a conventional nutrient medium, and the conventional nutrient medium is appropriately modified to induce a promoter, select a transformant, or amplify a gene encoding a necessary sequence.

[0237] The host cell for producing the antibody of the present application can be cultured in a plurality of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Patent Reissue 30,985 can be used as the medium for host cells. Any one of these media can be supplemented with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN (trademark) drug), trace elements (usually defined as inorganic compounds present at final concentrations within the micromolar range) and glucose or equivalent energy as needed. It may further contain any other necessary supplements at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those used with the host cell previously selected for expression and are obvious to those skilled in the art.

[0238] When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. When antibodies are produced intracellularly, as a first step, particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. When antibodies are secreted into the medium, usually, the supernatant from this expression system is first concentrated by a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis and may also contain antibiotics to prevent the growth of foreign contaminants.

[0239] Protein compositions produced from cells can be purified, for example, by hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, where affinity chromatography is the preferred purification technique. The matrix linked to the affinity ligand is usually agarose, but other matrices are also available. Compared to agarose, mechanically stable matrices, such as glass or poly(styrene - divinyl)benzene with controllable pore size, allow for faster flow rates and shorter processing times. Depending on the antibody to be recovered, other protein purification techniques, such as fractionation on an ion - exchange column, ethanol precipitation, reverse - phase HPLC, silica gel chromatography, heparin SEPHAROSE (trademark) chromatography on an anion or cation - exchange resin (e.g., polyaspartic acid column), chromatofocusing, SDS - PAGE, ammonium sulfate precipitation, may be used. After any one or more preliminary purification steps, low - pH hydrophobic interaction chromatography may be performed on the mixture containing the target antibody and contaminants.

[0240] 5.2.7. Binding Molecules Containing Single - Domain Antibodies In another aspect, the present specification provides a binding molecule comprising a single domain antibody according to the present specification (e.g., a VHH domain against claudin-6). In addition to the chimeric antigen receptor (CAR) according to the present specification described in Section 5.3 below, in some aspects, the single domain antibody according to the present specification against claudin-6 is also part of another binding molecule. Exemplary binding molecules of the present disclosure are described herein.

[0241] Fusion protein In various aspects, the single domain antibody according to the present specification can be fused or chemically conjugated to another agent, such as a protein-based entity. The single domain antibody can be chemically conjugated to the agent or conjugated to the agent in a non-covalent manner in other ways. The agent can be a peptide or an antibody (or a fragment thereof).

[0242] Therefore, in some aspects, the present specification provides a single domain antibody (e.g., a VHH domain) that recombinantly fuses or chemically conjugates (covalently or non-covalently conjugates) with a heterologous protein or polypeptide (or a fragment thereof, e.g., a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 amino acids or a polypeptide of more than 500 amino acids) to produce a fusion protein, and its use. Specifically, the present specification provides a fusion protein comprising an antigen-binding fragment (e.g., CDR1, CDR2, and / or CDR3) of the single domain antibody according to the present specification and a heterologous protein, polypeptide, or peptide.

[0243] In addition, the antibody according to the present specification can be fused with a label or "tag" sequence (e.g., a peptide) to facilitate purification. The label or tag amino acid sequence can be a hexahistidine peptide, a hemagglutinin ("HA") tag, and a "FLAG" tag.

[0244] Methods of fusing or conjugating a moiety (including a polypeptide) with an antibody are known (e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, Monoclonal Antibodies and Cancer Therapy 243-56 (edited by Reisfeld et al., 1985), Hellstrom et al., Antibodies for Drug Delivery, Controlled Drug Delivery 623-53 (edited by Robinson et al., 2nd ed., 1987), Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, Monoclonal Antibodies: Biological and Clinical Applications 475-506 (edited by Pinchera et al., 1985), Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, Monoclonal Antibodies for Cancer Detection and Therapy 303-16 (edited by Baldwin et al., 1985), Thorpe et al., Immunol. Rev. 62:119-58 (1982), U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946, EP 307434, EP 367166, EP 394827, PCT Publication WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813, Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-39 (1991), Traunecker et al., Nature, 331:84-86 (1988), Zheng et al., J. Immunol.See 154:5590-600(1995) and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-41(1992).

[0245] Fusion proteins can be produced, for example, by techniques collectively referred to as gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (“DNA shuffling”). DNA shuffling can be used to alter the activity of single domain antibodies according to the present specification, for example, including antibodies having high affinity and low dissociation rate (see, for example, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458, Patten et al., Curr. Opinion Biotechnol. 8:724-33(1997), Harayama, Trends Biotechnol. 16(2):76-82(1998), Hansson et al., J. Mol. Biol. 287:265-76(1999), and Lorenzo and Blasco, Biotechniques 24(2):308-13(1998)). Antibodies or the encoded antibodies can be altered by performing error-prone PCR, random nucleotide insertion or other methods to introduce random mutations prior to recombination. The polynucleotides encoding the antibodies according to the present specification can be recombined with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

[0246] Single domain antibodies according to the present specification (e.g., VHH domains) can be conjugated to a second antibody to form an antibody heteroconjugate.

[0247] In various embodiments, the single domain antibody is genetically fused to an effector. The genetic fusion can be achieved by placing a linker (e.g., a polypeptide) between the single domain antibody and the effector. The linker can be a flexible linker.

[0248] Single-domain antibodies can be conjugated to a therapeutic molecular gene, where the hinge region links the single-domain antibody to the therapeutic molecule.

[0249] This specification further provides methods for producing various fusion proteins according to this specification. The various methods described in Section 5.2.6 above can be used to produce the fusion proteins according to this specification.

[0250] In a specific embodiment, the fusion proteins according to this specification are recombined and expressed. Recombinant expression of the fusion proteins according to this specification may require the construction of an expression vector containing a polynucleotide encoding the protein or fragments thereof. When obtaining a polynucleotide encoding the protein or fragments thereof according to this specification, a vector for producing the molecule can be produced by recombinant DNA technology using techniques well known in the art. Therefore, this specification describes a method for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding it. Methods well known to those skilled in the art can be used to construct an expression vector containing a coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo gene recombination. A replicable vector is further provided, which contains a nucleotide sequence encoding a fusion protein according to this specification or fragments thereof or CDRs, operably linked to a promoter.

[0251] An expression vector can be transferred into a host cell by conventional techniques, and the transfected cells can be cultured by conventional techniques to produce the fusion proteins according to this specification. Therefore, this specification further provides a host cell containing a polynucleotide encoding a fusion protein according to this specification or fragments thereof, operably linked to a heterologous promoter.

[0252] Multiple host expression vector systems can be used to express the fusion proteins according to the present specification. Such host expression systems represent vehicles, the target coding sequences are produced by these vehicles and can then be purified, and together with cells, when transformed or transfected with appropriate nucleotide coding sequences, these cells can express the fusion proteins according to the present specification in situ. These include, but are not limited to, microorganisms, such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequence, yeast (e.g., Pichia) transformed with recombinant yeast expression vectors containing the coding sequence, insect cell lines infected with recombinant virus expression vectors (e.g., baculovirus), plant cell lines infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence, or mammalian cell lines (e.g., COS, CHO, BHK, 293, NS0 and 3T3 cells) containing recombinant expression constructs, and these recombinant expression constructs contain promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter). Bacterial cells such as Escherichia coli, or eukaryotic cells, especially bacterial cells for expressing complete recombinant antibody molecules, can be used to express recombinant fusion proteins. For example, the combination of mammalian cells such as Chinese hamster ovary cells (CHO) and vectors such as the major immediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies or their variants. In a specific embodiment, the expression of the nucleotide sequence encoding the fusion protein according to the present specification is regulated by a constitutive promoter, an inducible promoter or a tissue-specific promoter.

[0253] In the bacterial system, multiple expression vectors can be advantageously selected according to the desired use of the expressed fusion protein. For example, when it is necessary to produce such a fusion protein in large quantities, a vector that guides the expression of a high-level fusion protein product that is easy to purify may be required to produce a pharmaceutical composition of the fusion protein. These vectors include, but are not limited to, the Escherichia coli expression vector pUR278 (Ruther et al., EMBO 12:1791 (1983)), in which the coding sequence can be ligated alone to the vector together with the lac Z coding region in the framework, thereby producing the Escherichia coli expression vector pUR278 that produces the fusion protein, the pIN vector (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985), Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)), etc. The pGEX vector can also be used to express an exogenous polypeptide as a fusion protein with glutathione S-transferase (GST). Usually, such a fusion protein is soluble and can be easily purified from lysed cells by adsorbing and binding to matrix glutathione agarose beads and eluting in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site, whereby the cloned target gene product can be released from the GST moiety.

[0254] In mammalian host cells, many virus-based expression systems can be used. When adenovirus is used as an expression vector, the target coding sequence can be ligated to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. Then, the chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the virus genome (e.g., region E1 or E3) can produce a recombinant virus that is viable and can express the fusion protein in the infected host (see, for example, Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. Note that the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can have various sources, including natural and synthetic. Appropriate transcription enhancer elements, transcription terminators, etc. can be included to improve the expression efficiency (see, for example, Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0255] In addition, host cell lines can be selected that regulate the expression of the inserted array or modify and process gene products in a specific manner of the study. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms used for post-translational processing and modification of proteins and gene products. Selecting an appropriate cell line or host system can ensure the accurate modification and processing of the expressed exogenous protein. Therefore, eukaryotic host cells having appropriate cellular mechanisms for the processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.

[0256] To produce recombinant proteins in high yields over a long period of time, stable expression may be utilized. For example, cell lines that stably express a fusion protein may be manipulated. For using an expression vector containing a viral origin of replication, the host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selection marker. After introducing the exogenous DNA, the manipulated cells may be grown in enriched medium for 1 - 2 days and then transferred to selective medium. The selection marker in the recombinant plasmid confers resistance to selection, enables the cells to stably integrate the plasmid into their chromosomes and grow to form foci, and the foci can also be cloned and grown to become cell lines. The method can be advantageously used for the engineering of cell lines that express fusion proteins. Such manipulated cell lines are particularly useful for screening and evaluating compositions that interact directly or indirectly with the binding molecule.

[0257] Multiple selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine guanine phosphoribosyl transferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyl transferase (Lowy et al., Cell 22:8-17 (1980)). The genes can be used in tk-, hgprt-, or aprt- cells, respectively. Additionally, metabolic antagonist resistance can be used as the basis for the selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980), O’Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991), Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993), Mulligan, Science 260:926-932 (1993), and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993), May, TIB TECH 11(5):l55-2 15(1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods generally known in the field of recombinant DNA technology can be generally used for the selection of desired recombinant clones, and these methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990), and Dracopoli et al. (eds.), Current Protocols in Human Genetics, Chapters 12 and 13, John Wiley & Sons, NY (1994), Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), and these documents are hereby incorporated by reference in their entirety into this specification.

[0258] The expression level of the fusion protein can be increased by vector amplification (see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Volume 3 (Academic Press, New York, 1987) for a review). If the label in the vector system expressing the fusion protein is amplifiable, an increase in the inhibitor level present in the host cell culture causes an increase in the copy number of the labeled gene. Since the amplification region is related to the fusion protein gene, the production of the fusion protein also increases (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0259] Host cells can be co-transfected with multiple expression vectors according to the present specification. The vectors may contain the same selection markers, and these selection markers can equivalently express the corresponding encoded polypeptides. Alternatively, a single vector encoding and capable of expressing multiple polypeptides can be used. The coding sequence may include cDNA or genomic DNA.

[0260] When the fusion proteins according to the present specification are produced by recombinant expression, any method known in the art for purifying polypeptides (e.g., immunoglobulin molecules), such as chromatography (e.g., ion exchange, affinity, especially affinity for a specific antigen after protein A, size column chromatography, and Kappa select affinity chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins, can be used. In addition, the fusion protein molecules according to the present specification can be fused with heterologous polypeptide sequences described in the present specification or known in the art to facilitate purification.

[0261] Immunoconjugate The present disclosure further provides immunoconjugates, which include any of the antibodies described herein (e.g., anti-claudin-6 single domain antibodies) conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzyme-active toxins derived from bacteria, fungi, plants, or animals, or fragments thereof), or radioisotopes.

[0262] The immunoconjugate may be an antibody-drug conjugate (ADC), where the antibody is conjugated to one or more drugs, such as maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent EP 0 425 235 B1), auristatins, e.g., monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatin, calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296, Hinman et al., Cancer Res. 53:3336-3342 (1993), and Lode et al., Cancer Res. 58:2925-2928 (1998)), anthracyclines, e.g., daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006), Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006), Torgov et al., Bioconj. Chem. 16:717-721 (2005), Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000), Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002), King et al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Pat. No. 6,630,579), methotrexate, vindesine, taxanes, e.g., docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, trichothecenes, and CC1065, including but not limited to these.

[0263] The immunoconjugate may comprise an antibody described herein conjugated to an enzymatically active toxin or a fragment thereof, and the enzymatically active toxin or fragments thereof include, but are not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, midecamycin A chain, α-sarcin, abrin, dianthin, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, toxin, crocin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecin.

[0264] The immunoconjugate may comprise an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A plurality of radioisotopes may be used in the production of the radioactive conjugate. Examples include At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212 and radioisotopes of Lu. When the radioactive conjugate is used for detection, it may include a radioactive atom used in scintillation studies, such as tc99m or I123, or a spin label used in nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0265] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), diazo compounds (e.g., bis(p-azidobenzoyl)hexanediamine), diazonium derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate) and bis active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radio nucleotides and antibodies. See WO 94 / 11026.

[0266] The linker may be a "cleavable linker" that promotes release of the conjugate in the cell, but non-cleavable linkers are also contemplated herein. Linkers used in the conjugates of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide bond-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids, these amino acids, e.g., valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photo-labile linkers, dimethyl linkers, thioether linkers or hydrophilic linkers designed to avoid multi-drug transporter-mediated resistance.

[0267] The immunoconjugates or ADCs of this specification contemplate such conjugates manufactured using crosslinking reagent(s), but are not limited thereto, and these crosslinking reagent(s) are commercially available (e.g., obtained from Pierce Biotechnology, Inc., Rockford, IL, U.S.A.) and include, but are not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)vinyl sulfone).

[0268] In other embodiments, the antibodies according to this specification conjugate or recombinantly fuse, for example, with diagnostic molecules. Such diagnosis and detection can be accomplished, for example, by coupling the antibody with a detectable substance, and these detectable substances include, but are not limited to, various enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; linker groups such as streptavidin / biotin or avidin / biotin, but not limited thereto; fluorescent materials such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamide fluorescein, dansyl chloride, or phycoerythrin, but not limited thereto; luminescent materials such as luminol, but not limited thereto; bioluminescent materials such as luciferase, fluorescein, or aequorin, but not limited thereto; and chemiluminescent materials such as 225Acγ radiation, Auger radiation, β-radiation, α-radiation, or positron-emitting radioisotopes, but not limited thereto.

[0269] 5.3. Chimeric Antigen Receptor In another aspect, the present specification provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, the extracellular antigen-binding domain comprising a single-domain antibody (e.g., VHH) that binds to claudin-6 as described herein. Exemplary CARs (i.e., VHH-based CARs) comprising the VHH domain of the invention are described in Section 6 below.

[0270] In some aspects, a chimeric antigen receptor (CAR) according to the present specification comprises a polypeptide, the polypeptide comprising: (a) an extracellular antigen-binding domain comprising a single-domain antibody (sdAb) that specifically binds to claudin-6 as described herein and optionally one or more additional binding domains; (b) a transmembrane domain; and (c) an intracellular signaling domain. Each part and additional regions are described in more detail below.

[0271] 5.3.1. Extracellular antigen-binding domain The extracellular antigen-binding domain of the CARs described herein comprises one or more (e.g., any one of 1, 2, 3, 4, 5, 6 or more) single-domain antibodies. The single-domain antibodies can be directly fused to each other via a peptide bond or a peptide linker.

[0272] The CARs of the present disclosure comprise an extracellular antigen-binding domain, the extracellular antigen-binding domain comprising one or more single-domain antibodies. The sdAbs may have the same or different sources and may have the same or different sizes. In some aspects, the extracellular antigen-binding domain according to the present specification comprises at least one binding domain, and the at least one binding domain comprises a single-domain antibody that binds to claudin-6 as described herein, e.g., the anti-claudin-6 single-domain antibody described in Section 5.2 above.

[0273] In some embodiments, the present specification provides a CAR comprising a polypeptide, the polypeptide comprising: (a) an extracellular antigen-binding domain comprising an anti-claudin-6 sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-claudin-6 sdAb is the anti-claudin-6 sdAb described in Section 5.2 above.

[0274] In some embodiments, the present specification provides a CAR comprising a polypeptide, the polypeptide comprising (a) an extracellular antigen-binding domain comprising an anti-claudin-6 sdAb, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the anti-claudin-6 sdAb comprises (i) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:7, (ii) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:8, (iii) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:9, (iv) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:10, or (v) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:11. In some embodiments, CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In some embodiments, the anti-claudin-6 sdAb comprises (i) CDR1 comprising the amino acid sequence of SEQ ID NO:1, CDR2 comprising the amino acid sequence of SEQ ID NO:3, and CDR3 comprising the amino acid sequence of SEQ ID NO:5, or (ii) CDR1 comprising the amino acid sequence of SEQ ID NO:2, CDR2 comprising the amino acid sequence of SEQ ID NO:4, and CDR3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the anti-claudin-6 sdAb comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.In some embodiments, the anti-claudin-6 sdAb comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

[0275] In other embodiments, the extracellular antigen-binding domain comprises two or more antigen-binding domains. In these two or more antigen-binding domains, at least one is a VHH that binds to claudin-6 according to the present specification, and one or more additional binding domains that bind to one or more additional antigens, for example, one, two, three, four or more additional single-domain antibody binding regions (sdAbs) that target one or more additional antigens. In some embodiments, at least one of these additional binding domains binds to GPC3. In some specific embodiments, the additional binding domain comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 18 and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 19. In some embodiments, CDR1, CDR2 or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme or a combination thereof. In other embodiments, the additional binding domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 14 and HCDR3 containing the amino acid sequence of SEQ ID NO: 16, and LCDR1 containing the amino acid sequence of SEQ ID NO: 13, LCDR2 containing the amino acid sequence of SEQ ID NO: 15 and LCDR3 containing the amino acid sequence of SEQ ID NO: 17. In some embodiments, the additional binding domain comprises a VH domain containing SEQ ID NO: 18 and a VL domain containing SEQ ID NO: 19.In some embodiments, the additional binding domain comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:18, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:19. In some embodiments, the additional binding domain is a single-chain antibody fragment (scFv). In some embodiments, the additional antigen-binding domain comprises the amino acid sequence of SEQ ID NO:20. In some embodiments, the additional antigen-binding domain comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of SEQ ID NO:20. In some embodiments, the antigen-binding domains are fused to each other via a peptide linker.

[0276] In addition to one or more antigen-binding domains according to the present specification, the CAR according to the present specification may further comprise one or more of a linker (e.g., a peptide linker), a transmembrane domain, a hinge region, a signal peptide, an intracellular signaling domain, a co-stimulatory signaling domain, which will be described in more detail below, respectively.

[0277] For example, in some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand of CD83, and combinations thereof. In some embodiments, the co-stimulatory signaling domain is derived from CD137. In some embodiments, the Claudin-6 CAR further comprises a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the Claudin-6 CAR further comprises a signal peptide (e.g., a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the Claudin-6 CAR is monospecific. In some embodiments, the Claudin-6 CAR is monovalent. In some embodiments, the Claudin-6×GPC3 CAR according to the present specification is bispecific.

[0278] Peptide linker When multiple antibodies (e.g., multiple antibody fragments) are present in the CAR of the present invention, each antibody can be fused to each other via a peptide linker. Antibodies can be fused directly to each other in the absence of any peptide linker. The peptide linkers connecting different antibodies may be the same or different. Different domains of the CAR can also be fused to each other via a peptide linker.

[0279] Depending on the structure and / or functional characteristics of the antibody and / or each domain, each peptide linker in the CAR may have the same or different lengths and / or sequences. Each peptide linker can be independently selected and optimized. The length, flexibility and / or other properties of one or more peptide linkers used in the CAR may have some impact on the properties, including but not limited to the affinity, specificity or avidity for one or more specific antigens or epitopes. For example, a longer peptide linker may be selected to ensure that two adjacent domains do not spatially interfere with each other. A short peptide linker may be placed between the transmembrane domain and the intracellular signaling domain of the CAR. The peptide linker may contain flexible residues (such as glycine and serine), whereby the adjacent domains can move freely relative to each other. For example, a glycine-serine dimer may be a suitable peptide linker.

[0280] The peptide linker may have any suitable length. The length of the peptide linker may be any one of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. The length of the peptide linker may be any one or less of about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. The length of the peptide linker may be any one of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids or about 1 amino acid to about 100 amino acids.

[0281] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, the sequence of the hinge region derived from an antibody having only a heavy chain may be used as a linker. See, for example, WO1996 / 34103. The peptide linker may be a flexible linker. Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Other linkers known in the art may be included in the CARs according to the present specification as described in, for example, WO 2016014789, WO 2015158671, WO 2016102965, US 20150299317, WO 2018067992, US7741465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1**9**0) and Bird et al., Science 242:423-426 (1988), and each disclosure is incorporated herein by reference.

[0282] In some specific embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41.

[0283] 5.3.2. Transmembrane domain The CARs of the present disclosure include a transmembrane domain that can be fused directly or indirectly to the extracellular antigen-binding domain. The transmembrane domain may be derived from a natural or synthetic source. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, such as a eukaryotic cell membrane. Transmembrane domains suitable for the CARs described herein can be obtained from naturally occurring proteins. Alternatively, it may be a synthetic, non-natural protein segment, such as a hydrophobic segment that is thermodynamically stable in a cell membrane.

[0284] Classify transmembrane domains based on their three-dimensional structures. For example, transmembrane domains can form α-helices, complexes of one or more α-helices, β-barrels, or any other stable structure that can span the cell lipid bilayer. Additionally, transmembrane domains can be further classified, either similarly or alternatively, based on the transmembrane domain topology structure that includes the number of times the transmembrane domain crosses the membrane and the orientation of the protein. For example, single-pass transmembrane proteins cross the cell membrane once, while multi-pass transmembrane proteins cross the cell membrane at least 2 (e.g., 2, 3, 4, 5, 6, 7, or more) times. Membrane proteins can be defined as type I, type II, or type III depending on the topology structure of their termini and one or more transmembrane segments with respect to the inside and outside of the cell. Type I membrane proteins have a single transmembrane region and are oriented such that the N-terminus of the protein is located extracellularly to the lipid bilayer of the cell and the C-terminus of the protein is present intracellularly. Type II membrane proteins also have a single transmembrane region but are oriented such that the C-terminus of the protein is present extracellularly to the lipid bilayer of the cell and the N-terminus of the protein is present intracellularly. Type III membrane proteins have multiple transmembrane segments and can be further classified based on the number of transmembrane segments and the positions of the N-terminus and C-terminus.

[0285] The transmembrane domain of the CAR described herein may be derived from a type I single-pass transmembrane protein. Transmembrane domains from multi-pass transmembrane proteins may be used to be compatible with the CAR described herein. Multi-pass transmembrane proteins may include complexes (at least 2, 3, 4, 5, 6, 7, or more) of α-helices or β-sheet structures. The N-terminus and C-terminus of multi-pass transmembrane proteins may be present on opposite sides of the lipid bilayer. For example, the N-terminus of the protein is present intracellularly to the lipid bilayer, while the C-terminus of the protein is present extracellularly.

[0286] The transmembrane domain of CAR may include a transmembrane domain selected from the transmembrane domains of the α, β, or ζ chains of the T cell receptor, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, Claudin-6, IL-2R β, IL-2R γ, IL-7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0287] In some specific embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain is the transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO:36.

[0288] The transmembrane domain used in the CAR described herein may further comprise at least a portion of a synthetic, non-natural protein segment. The transmembrane domain may be a synthetic, non-natural alpha helix or beta sheet. The protein segment may be at least about 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Examples of synthetic transmembrane domains are known in the art and are described, for example, in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0289] The transmembrane domain according to the present specification may include a transmembrane region and an intracellular region located on the C-terminal side of the transmembrane domain. The intracellular region of the transmembrane domain may contain three or more amino acids and, in some embodiments, is advantageous for the orientation of the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. One or more cysteine residues may be present in the intracellular region of the transmembrane domain. The intracellular region of the transmembrane domain may contain positively charged amino acids. The intracellular region of the transmembrane domain may contain the amino acids arginine, serine, and lysine.

[0290] The transmembrane region of the transmembrane domain may contain hydrophobic amino acid residues. The transmembrane domain of the CAR according to the present specification may contain a hydrophobic artificial sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminal position of the transmembrane domain. The transmembrane region may mainly contain hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. The transmembrane region may be hydrophobic. The transmembrane region may contain a poly-leucine-alanine sequence. The hydrophilic or hydrophobic or hydrophilic characteristics of a protein or protein segment can be evaluated by any method known in the art, such as Kyte and Doolittle hydrophilicity analysis.

[0291] 5.3.3. Intracellular Signaling Domain The CARs of the present disclosure include an intracellular signaling domain. The intracellular signaling domain serves to activate at least one normal effector function of the immune effector cells expressing the CAR. The term "effector function" refers to a specific function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the protein portion that transmits an effector function signal and instructs the cell to perform a specific function. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. Regarding the use of a shortened portion of the intracellular signaling domain, such a shortened portion may be used instead of the intact chain as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is intended to refer to any shortened portion that includes an intracellular signaling domain sufficient to transmit an effector function signal.

[0292] In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of the primary intracellular signaling domain of an immune effector cell. The "primary intracellular signaling domain" refers to an intracellular signaling sequence that acts stimulatorily to induce an immune effector to function. The primary intracellular signaling domain may contain an immunoreceptor tyrosine-based activation motif or a signaling motif called an immunoreceptor tyrosine-based activation motif (ITAM). As used herein, "ITAM" is a conserved protein motif that is typically present in the tail of signaling molecules expressed in immune cells. The motif may include two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. The ITAM in a signaling molecule is important for intracellular signaling, which is at least partly mediated by phosphorylation of tyrosine residues in the ITAM after activation of the signaling molecule. The ITAM can also function as a docking site for other proteins involved in the signaling pathway. Exemplary primary intracellular signaling sequences containing ITAM include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0293] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. The intracellular signaling domain may be composed of the intracellular signaling domain of CD3ζ. The primary intracellular signaling domain may be the intracellular signaling domain of wild-type CD3ζ. In some embodiments, the primary intracellular signaling domain of CD3ζ comprises the amino acid sequence of SEQ ID NO:38.

[0294] 5.3.4. Co-stimulatory signaling domain In addition to stimulating antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival and to activate the effector functions of the cells. In some embodiments, the CAR comprises at least one co-stimulatory signaling domain. As used herein, the term "co-stimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signaling to induce an immune response (e.g., effector function). The co-stimulatory signaling domain of the chimeric receptor described herein may be an intracellular signaling domain from a co-stimulatory protein, which transmits signals and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "co-stimulatory signaling domain" may be the intracellular portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognate binding partner in an immune cell (e.g., a T cell), which mediates a co-stimulatory response of the immune cell, such as, but not limited to, proliferation and survival, by specifically binding to a co-stimulatory ligand.

[0295] In some embodiments, the intracellular signaling domain comprises a single co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (e.g., any one of about 2, 3, 4 or more) co-stimulatory signaling domains. The intracellular signaling domain may comprise two or more identical co-stimulatory signaling domains. The intracellular signaling domain may comprise two or more co-stimulatory signaling domains from different co-stimulatory proteins (e.g., any two or more of the co-stimulatory proteins described herein). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3ζ) and one or more co-stimulatory signaling domains. The one or more co-stimulatory signaling domains and the primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3ζ) can be fused to each other via an optional peptide linker. The primary intracellular signaling domain and the one or more co-stimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3ζ). The plurality of co-stimulatory signaling domains can provide an additive or synergistic stimulatory effect.

[0296] Activation of costimulatory signaling domains in host cells (e.g., immune cells) can direct the cells to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domains of any costimulatory molecule are applicable to the CARs described herein. One or more types of costimulatory signaling domains are selected based on factors such as the type of immune effector cell in which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effector). Examples of costimulatory signaling domains used in CARs may be the intracellular signaling domains of costimulatory proteins, members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6), and members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNFRII / TNFRSF1B), members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150), and any other costimulatory molecules, such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C, but not limited thereto.

[0297] In some embodiments, one or more costimulatory signaling domains are selected from the group consisting of ligands that specifically bind to CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

[0298] In some embodiments, the intracellular signaling domain in the CAR of the present disclosure includes a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain includes the intracellular signaling domain of CD3ζ and the costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain includes the costimulatory signaling domain of CD137, which includes the amino acid sequence of SEQ ID NO:37.

[0299] Variants of any co-stimulatory signaling domain described herein are also within the scope of the present disclosure, whereby the co-stimulatory signaling domain can regulate the immune response of immune cells. Compared to wild-type counterparts, the co-stimulatory signaling domain may contain up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residue mutations. Such co-stimulatory signaling domains containing one or more amino acid changes may be referred to as variants. Compared to the co-stimulatory signaling domain without mutations, mutations in the amino acid residues of the co-stimulatory signaling domain can result in an increase in signal transduction and an enhancement of the stimulation to the immune response. Compared to the co-stimulatory signaling domain without mutations, mutations in the amino acid residues of the co-stimulatory signaling domain can result in a decrease in signal transduction and a reduction in the stimulation to the immune response.

[0300] 5.3.5. Hinge Region The CARs of the present disclosure may include a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. The hinge domain is typically an amino acid segment that is expressed between two domains of a protein and can enable the relative movement of one or two flexible domains of the protein with respect to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain with respect to the transmembrane domain of the effector molecule can be used.

[0301] The hinge domain may contain any one of about 10 to 100 amino acids, such as about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. The length of the hinge domain may be at least any one of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

[0302] The hinge domain may be the hinge domain of a naturally occurring protein. The hinge domain of any known protein in the art that contains a hinge domain is applicable to the chimeric receptor described herein. The hinge domain may be at least a part of the hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. The hinge domain may be a part of the hinge domain of CD8α, for example, containing a fragment of at least 15 (such as 20, 25, 30, 35 or 40) consecutive amino acids of the hinge domain of CD8α. In some embodiments, the hinge domain of CD8α comprises the amino acid sequence of SEQ ID NO:35.

[0303] The hinge domain of an antibody (e.g., IgG, IgA, IgM, IgE or IgD antibody) is also applicable to the pH-dependent chimeric receptor system described herein. The hinge domain may be the hinge domain that links the constant domains CH1 and CH2 of the antibody. The hinge domain may be the hinge domain of the antibody and includes the hinge domain of the antibody and one or more constant regions of the antibody. The hinge domain may include the hinge domain of the antibody and the CH3 constant region of the antibody. The hinge domain may include the hinge domain of the antibody and the CH2 and CH3 constant regions of the antibody. The antibody may be an IgG, IgA, IgM, IgE, or IgD antibody. Optionally, the antibody is an IgG antibody. The antibody may be an IgG1, IgG2, IgG3 or IgG4 antibody. The hinge region may include the hinge region of the IgG1 antibody and the CH2 and CH3 constant regions. The hinge region may include the hinge region of the IgG1 antibody and the CH3 constant region.

[0304] Non-natural peptides may be used as the hinge domain of the chimeric receptors described herein. The hinge domain between the C-terminus of the extracellular ligand-binding domain of the Fc receptor and the N-terminus of the transmembrane domain may be a peptide linker, for example, a (GxS)n linker, where x and n may independently be an integer from 3 to 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.

[0305] 5.3.6. Signal Peptide The CARs of the present disclosure may include a signal peptide (also referred to as a signal sequence) at the N-terminal position of the polypeptide. Generally, a signal peptide is a peptide sequence that targets the polypeptide to a desired site within the cell. The signal peptide can target effector molecules to the secretory pathway of the cell and enable the incorporation and anchoring of the effector molecules into the lipid bilayer. It will be apparent to those skilled in the art which signal peptides, including signal sequences of naturally occurring proteins or synthetic non-natural signal sequences, are applicable to the CARs described herein. The signal peptide may be derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is from CD8α. In some embodiments, the signal peptide of CD8α comprises the amino acid sequence of SEQ ID NO:34.

[0306] 5.3.7. Exemplary CARs that Bind Claudin-6 and / or GPC3 Exemplary CARs that bind to Claudin-6 are generated as shown in Section 6 below and are, for example, CNBCAR1. In some embodiments, the present specification provides a CAR comprising or consisting of the amino acid sequence of SEQ ID NO:26. In some embodiments, the CAR according to the present specification comprises an amino acid sequence having a certain percentage identity to any one of the exemplary CARs in Section 6 below. In some embodiments, the present specification provides a Claudin-6 CAR comprising a peptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:26.

[0307] In some embodiments, the present specification provides a nucleic acid encoding any Claudin-6 CAR according to the present specification. More detailed descriptions regarding nucleic acid sequences and vectors are provided below.

[0308] Exemplary CARs that bind to Claudin-6 and GPC3 are generated as shown in Section 6 below and are, for example, CNBCAR3. In some embodiments, the present specification provides a CAR comprising or consisting of the amino acid sequence of SEQ ID NO:28. In some embodiments, the CAR according to the present specification comprises an amino acid sequence having a certain percentage identity to any one of the exemplary CARs in Section 6 below. In some embodiments, the present specification provides a Claudin-6×GPC3 CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:28.

[0309] In some embodiments, the present specification provides a nucleic acid encoding any Claudin-6×GPC3 CAR according to the present specification. More detailed descriptions regarding nucleic acid sequences and vectors are provided below.

[0310] Other exemplary CARs according to the present specification further include chimeric receptors. In some embodiments, the chimeric receptor includes TGFβR and / or IL23R. The CAR according to the present specification may include the amino acid sequence of SEQ ID NO:29. The CAR according to the present specification may include the amino acid sequence of SEQ ID NO:31. The present specification can provide a CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:29. The present specification can provide a CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:31.

[0311] In some embodiments, the present specification provides a nucleic acid encoding any CAR and chimeric receptor according to the present specification. More detailed descriptions regarding nucleic acid sequences and vectors are provided below.

[0312] 5.4. Engineered immune effector cells In another embodiment, the present specification provides a host cell (e.g., an immune effector cell) comprising any one of the CARs described herein.

[0313] Thus, in some embodiments, the present specification includes engineered immune effector cells (e.g., T cells) comprising a CAR, the CAR comprising a polypeptide, the polypeptide comprising (a) an extracellular antigen-binding domain comprising one or more anti-claudin-6 sdAbs, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the anti-claudin-6 sdAb is the anti-claudin-6 sdAb described in Section 5.2 above, e.g., (i) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:7, (ii) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:8, (iii) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:9, (iv) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:10, or (v) CDR1, CDR2, and CDR3 each having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:11. In some embodiments, CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In some embodiments, the anti-claudin-6 sdAb comprises (i) CDR1 comprising the amino acid sequence of SEQ ID NO:1, CDR2 comprising the amino acid sequence of SEQ ID NO:3, and CDR3 comprising the amino acid sequence of SEQ ID NO:5, or (ii) CDR1 comprising the amino acid sequence of SEQ ID NO:2, CDR2 comprising the amino acid sequence of SEQ ID NO:4, and CDR3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the anti-claudin-6 sdAb comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.In some embodiments, the anti-claudin-6 sdAb comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

[0314] In some embodiments, the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains. In some embodiments, the additional binding domains comprise HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:18 and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:19. In some embodiments, CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In other embodiments, the additional binding domains comprise HCDR1 containing the amino acid sequence of SEQ ID NO:12, HCDR2 containing the amino acid sequence of SEQ ID NO:14, HCDR3 containing the amino acid sequence of SEQ ID NO:16, and LCDR1 containing the amino acid sequence of SEQ ID NO:13, LCDR2 containing the amino acid sequence of SEQ ID NO:15, and LCDR3 containing the amino acid sequence of SEQ ID NO:17. In some embodiments, the additional binding domains comprise a VH domain containing SEQ ID NO:18 and a VL domain containing SEQ ID NO:19. In some embodiments, the additional binding domains comprise a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:18 and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:19. In some embodiments, the additional binding domain is a single-chain antibody fragment (scFv). In some embodiments, the additional antigen-binding domain comprises the amino acid sequence of SEQ ID NO:20.In some embodiments, the additional antigen-binding domain comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of SEQ ID NO:20. In some embodiments, the antigen-binding domains are fused to each other via a peptide linker. In some embodiments, the length of the peptide linker is 50 amino acids or less. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of CD83 and combinations thereof. In some embodiments, the CAR further comprises a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the CAR further comprises a signal peptide (e.g., a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signaling domain derived from CD137 and a primary intracellular signaling domain derived from CD3ζ.

[0315] In other specific embodiments, the present specification provides engineered immune effector cells (e.g., T cells) comprising a CAR, the CAR comprising a polypeptide, the polypeptide comprising the amino acid sequence of SEQ ID NO: 26 or 28, or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26 or 28.

[0316] In some embodiments, the engineered immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells or embryonic stem cells. The T cells may be αβ T cells or γδ T cells. The engineered immune effector cells may be autologous. The engineered immune effector cells may be allogeneic.

[0317] The engineered immune effector cells can further express one or more therapeutic proteins and / or immunomodulators, such as immune checkpoint inhibitors.

[0318] 5.4.2. Vector The present disclosure provides a vector for cloning and expressing any one of the CARs described herein. The vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. The vector may be a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex viral vectors and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0319] Many virus-based systems have been developed for introducing genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using known techniques in the art, heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles. Then, recombinant viruses can be isolated and delivered to engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. Adenoviral vectors may be used. Many adenoviral vectors are known in the art. Lentiviral vectors may be used. Self-inactivating lentiviral vectors may be used. For example, self-inactivating lentiviral vectors carrying an immunomodulatory agent (e.g., an immune checkpoint inhibitor) coding sequence and / or self-inactivating lentiviral vectors carrying a chimeric antigen receptor can be packaged with known regimens in the art. Using known methods in the art, the resulting lentiviral vectors may be used for transduction of mammalian cells (e.g., primary human T cells). Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for long-term and stable integration of transgenes and proliferation in progeny cells. Lentiviral vectors also have low immunogenicity and can be transduced into non-proliferating cells.

[0320] In some embodiments, the vector comprises any one of the nucleic acids encoding a CAR described herein. The nucleic acid can be cloned into the vector using any well-known molecular cloning method in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. The nucleic acid can be operably linked to a promoter. Various promoters for gene expression in mammalian cells have been explored, and any promoter known in the art can be used in the present disclosure. Promoters can be broadly classified into constitutive promoters or regulatable promoters, e.g., inducible promoters.

[0321] The nucleic acid encoding the CAR can be operably linked to a constitutive promoter. A constitutive promoter enables constitutive expression of a heterologous gene (also called transgenic) in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the cytomegalovirus virus (CMV) promoter, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerate kinase promoter (PGK), simian virus 40 early promoter (SV40), and the chicken β-actin promoter (CAGG) coupled to the CMV early enhancer. The efficiency of such constitutive promoters for driving transgenic expression has been widely compared in many studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1α, UbiC, and PGK to drive chimeric antigen receptor expression in primary human T cells and concluded that the hEF1α promoter not only induces the highest level of transgenic expression but is also optimally maintained in CD4 and CD8 human T cells (Molecular Therapy, 17(8): 1453-1464 (2009)). The nucleic acid encoding the CAR can be operably linked to the hEF1α promoter.

[0322] The nucleic acid encoding the CAR can be operably linked to an inducible promoter. Inducible promoters belong to the class of regulatable promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the engineered immune effector cells or the physiological state of the engineered immune effector cells, inducers (i.e., inducing agents), or combinations thereof.

[0323] The inducing conditions may not induce the expression of endogenous genes in the engineered mammalian cells and / or the subject receiving the pharmaceutical composition. The inducing conditions can be selected from the group consisting of inducers, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and the activation state of the engineered mammalian cells.

[0324] The vector may further comprise a selection marker gene or a reporter gene for selecting cells expressing the CAR from a group of host cells transfected with a lentiviral vector. To enable expression in the host cell, both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences. For example, the vector contains transcription and translation terminators, an initiation sequence, and a promoter that can be used to regulate the expression of the nucleic acid sequence.

[0325] The vector may contain one or more nucleic acids encoding the CAR. The vector may contain a nucleic acid containing a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first nucleic acid is functionally linked to the second nucleic acid via a third nucleic acid sequence encoding a self-cleaving peptide. The self-cleaving peptide may be selected from the group consisting of T2A, P2A, and F2A.

[0326] 5.4.3. Immune effector cells "Immune effector cells" refers to immune cells that can exert immune effector functions. Immune effector cells express at least FcγRIII and can exert an ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0327] In some embodiments, the immune effector cell is a T cell. The T cell may be an αβ T cell or a γδ T cell. The T cell may be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8- or a combination thereof. After expressing the CAR and binding to a target cell (e.g., a GPC3+ tumor cell), the T cell can produce IL-2, TFN, and / or TNF. CD8+ T cells can lyse antigen-specific target cells after expressing the CAR and binding to the target cells.

[0328] In some embodiments, the immune effector cells are NK cells. The immune effector cells may be established cell lines, such as NK-92 cells.

[0329] In some embodiments, the immune effector cells are differentiated from stem cells (e.g., hematopoietic stem cells, pluripotent stem cells, iPS, or embryonic stem cells).

[0330] Engineered immune effector cells are produced by introducing a CAR into the immune effector cells, such as T cells. In some embodiments, the CAR is introduced into the immune effector cells by transfecting any one of the nucleic acids or any one of the above vectors. The CAR can be introduced into the immune effector cells by inserting the protein into the cell membrane and passing the cells through a microfluidic system (e.g., CELL SQUEEZE (registered trademark)) (see, for example, US Patent Application Publication No. 20140287509).

[0331] Methods for introducing a vector or isolated nucleic acid into mammalian cells are known in the art. The vectors described can be transferred into immune effector cells by physical, chemical, or biological methods.

[0332] Physical methods for introducing a vector into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The vector can be introduced into the cells by electroporation.

[0333] Biological methods for introducing vectors into immune effector cells include using DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.

[0334] Chemical methods for introducing vectors into immune effector cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems such as water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0335] RNA molecules encoding any of the CARs described herein can be produced by conventional methods (e.g., in vitro transcription) and introduced into immune effector cells by known methods such as mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17:1027-1035 (2006).

[0336] In some embodiments, transduced or transfected immune effector cells are expanded ex vivo after introduction into the vector or nucleic acid. The transduced or transfected immune effector cells can be cultured and expanded for any of at least about 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days. The transduced or transfected immune effector cells can be further evaluated or screened to select the engineered mammalian cells.

[0337] Reporter genes can be used to identify transfectable cells and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that encodes a polypeptide that is not present in or not expressed in the recipient organism or tissue and that is indicated by a property (e.g., enzymatic activity) that can be easily detected for its expression. Expression of the reporter gene is detected at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and can be produced using known techniques or obtained commercially.

[0338] Other methods for confirming the presence of a nucleic acid encoding a CAR in engineered immune effector cells include, for example, molecular biology assays well known to those skilled in the art, such as Northern blotting and Southern blotting, RT-PCR and PCR, and biochemical assays that detect the presence or absence of specific peptides by immunological methods (e.g., ELISA and Western blot).

[0339] 5.4.4. Source of T cells Before performing amplification and genetic modification on T cells, a source of T cells can be obtained from a subject. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. Any number of T cell lines available in the art can be used. T cells can be obtained from a unit of blood collected from a subject using various techniques well known to those skilled in the art (e.g., Ficoll™ isolation). Cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes and include T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma portion and used in subsequent processing steps in an appropriate buffer or medium. The cells are washed with phosphate-buffered saline (PBS). The wash solution may lack calcium and may lack magnesium, or may lack many (but not all) divalent cations. The initial activation step in the absence of calcium can lead to an expansion of activation. As will be readily understood by those skilled in the art, the washing step can be completed by methods known in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca 2+ , Mg 2+ -free PBS, PlasmaLyte A, or other aqueous saline solutions with or without buffers. Alternatively, unwanted components of the apheresis sample can be removed and the cells can be resuspended directly in the medium.

[0340] For example, red blood cells can be lysed and monocytes depleted by PERCOLL (trademark) gradient centrifugation or countercurrent centrifugal elutriation to isolate T cells from peripheral blood lymphocytes. Specific subsets of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, T cells are isolated by incubating with anti-CD3 / anti-CD28 (i.e., 3×28) conjugation beads (e.g., DYNABEADS (registered trademark) M-450 CD3 / CD28 T) for a time sufficient to perform positive selection on the desired T cells. This period may be about 30 minutes. The range of this period may be from 30 minutes to 36 hours or more, and all integer values in between. This period may be at least 1, 2, 3, 4, 5 or 6 hours. This period may be from 10 to 24 hours. The incubation period may be 24 hours. In the isolation of T cells from leukemia patients, the cell yield can be increased by taking a longer incubation time (e.g., 24 hours). In any case where there are few T cells, such as when obtaining tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals compared to other cell types, T cells may be isolated with a long incubation time. Additionally, by using a long incubation, the capture efficiency of CD8+ T cells can be improved. Therefore, in some embodiments, by simply shortening or lengthening the time allowing binding of T cells to CD3 / CD28 beads and / or increasing or decreasing the ratio of beads to T cells, subsets of T cells can be preferentially selected or not selected at the start of culture or at other points during culture. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subsets of T cells can be preferentially selected or not selected at the start of culture or at other desired points in time. Those skilled in the art will recognize that multiple selection rounds can also be used. It may also be desirable to perform the selection procedure and use the "unselected" cells in the activation and amplification process."Unselected" cells can also undergo further rounds of selection.

[0341] Enrichment of a T cell population by negative selection can be achieved by binding with an antibody against a surface marker specific to the negatively selected cells. One method is to perform cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. It may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, deplete regulatory T cells by anti-C25 conjugated beads or other similar selection methods.

[0342] To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) may be varied. In order to ensure that the contact between the cells and the beads is maximized, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the increase of cells). For example, a concentration of 2 billion cells / ml is used. A concentration of 1 billion cells / ml may be used. A concentration of more than 100 million cells / ml may be used. Concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml may be used. Concentrations of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml may be used. Concentrations of 125 million or 150 million cells / ml may be used. The use of high concentrations can result in an increase in cell yield, cell activation and cell proliferation. Note that the use of high concentrations of cells can enable more effective capture of cells that may weakly express the target antigen of interest (e.g., CD28-negative T cells), or cells from samples in which many tumor cells are present (i.e., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are expected to be obtained. For example, the use of high cell concentrations enables more effective selection of CD8+ T cells that usually have weak CD28 expression.

[0343] It may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surfaces (e.g., particles such as beads), the interaction between the particles and the cells is minimized. Thereby, cells that express the desired antigen in high amounts to bind to the particles are selected. For example, CD4+ T cells express a higher level of CD28 and are captured more effectively than CD8+ T cells at diluted concentrations. The concentration of cells used is 5×10 6 cells / mL. The concentration used can be about 1×10 5 cells / mL to 1×10 6 cells / mL, and any integer value in between.

[0344] The cells can be incubated on a rotator at 2°C to 10°C or at room temperature for various lengths of time at various speeds.

[0345] T cells for stimulation may be frozen after the washing step. Without being bound by theory, the freezing and subsequent thawing steps can provide a more homogeneous product by removing granulocytes and to some extent monocytes in the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and are useful in such cases, but one method is PBS containing 20% DMSO and 8% human serum albumin, or 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or a medium containing 31.25% plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A. And it relates to freezing the cells at -80°C at a rate of 1°C per minute and storing them in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods and uncontrolled immediate freezing at -20°C or in liquid nitrogen can be used.

[0346] As described herein, the cryopreserved cells can be thawed, washed, and allowed to stand at room temperature for one hour before activation.

[0347] In the present disclosure, it is also contemplated to collect a blood sample or an apheresis product from a subject at a time point prior to when the amplified cells described herein may be needed. Thus, the cell source for amplification can be collected at any necessary time point, the necessary cells such as T cells can be isolated and frozen, and thereby later in T cell therapy, can be used for various diseases or conditions that benefit from T cell therapy as described herein. The blood sample or apheresis can be taken from a general healthy subject. The blood sample or apheresis blood components can be taken from a general healthy subject who is at risk of developing a disease but has not yet developed the disease, and the target cells are isolated and frozen for later use. T cells can be amplified, frozen, and used later. In some embodiments, the sample is taken from the patient immediately after diagnosis of a particular disease as described herein but prior to any treatment. Cells are isolated from the subject's blood sample or apheresis blood components prior to various related treatment modalities, and these related treatment modalities include, but are not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies or other immunoablative agents (e.g., CAMPATH, anti-CD3 antibody, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228) and radiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or the p70S6 kinase (rapamycin) that is important for growth factor-induced signal transduction (Liu et al., Cell 66:807-815 (1991), Henderson et al., Immun 73:316-321 (1991), Bierer et al., Curr. Opin. Immun. 5:763-773 (1993)).The patient's cells can be isolated and later frozen for use in combination with (e.g., before, simultaneously with, or after) bone marrow or stem cell transplantation, T cell depletion therapy using chemotherapeutic agents (e.g., fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or antibodies (e.g., OKT3 or CAMPATH).

[0348] T cells can be obtained directly from the patient after treatment. In this regard, after some cancer treatments, particularly after treatment with drugs that damage the immune system, the quality of the T cells obtained can be optimal, or their ex vivo proliferative capacity can be observed to improve, during the period when the patient would normally be recovering from the treatment, immediately after the treatment. Similarly, after ex vivo manipulation by the methods described herein, these cells can be in a favorable state for engraftment and in vivo amplification. Thus, in the context of the present disclosure, it is contemplated to collect blood cells during this recovery period, including T cells, dendritic cells, or other hematopoietic cells. Mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in the subject, where they are favorable for repopulation, recirculation, regeneration, and / or amplification of cell types, particularly during a defined time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0349] 5.4.5.Activation and Amplification of T Cells In some embodiments, before or after genetic modification of the T cells having the CARs described herein, the T cells can be activated and amplified, typically using methods described in the documents such as U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0350] Normally, T cells can be amplified by contacting with a surface to which a reagent that stimulates CD3 / TCR complex-related signals and a ligand that stimulates co-stimulatory molecules on the T cell surface are attached. Specifically, as described herein, a group of T cells can be stimulated, for example, by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof or an anti-CD2 antibody immobilized on the surface, or by contacting with a protein kinase C activator (e.g., bryostatin) that binds to a calcium ionophore. To co-stimulate helper molecules on the T cell surface, a ligand that binds to the helper molecule may be used. For example, under conditions suitable for stimulating the proliferation of T cells, a group of T cells may be contacted with an anti-CD3 antibody and an anti-CD28 antibody. Anti-CD3 antibodies and anti-CD28 antibodies may be used to stimulate the proliferation of CD4+ T cells or CD8+ T cells. Examples of anti-CD3 antibodies include UCHT1, OKT3, HIT3a (BioLegend, San Diego, US), and may be used as in other methods well known in the art (Graves J et al., J. Immunol. 146:2102 (1991), Li B et al., Immunol. 116:487 (2005), Rivollier A et al., Blood 104:4029 (2004)). Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), and may be used as in other methods well known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, (1998), Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999, Garland et al., J. Immunol Meth. 227(1-2):53-63 (1999)).

[0351] Primary and costimulatory signals for T cells can be provided by various regimens. For example, the agents providing each signal can be in solution or coupled to a surface. When coupled to a surface, the agents can be coupled to the same surface (i.e., in the "cis" format) or to separate surfaces (i.e., in the "trans" format). Alternatively, one agent can be coupled to a surface and another agent in solution. Reagents providing costimulatory signals can bind to the cell surface, and reagents providing primary activation signals can be in solution or coupled to a surface. Both agents can be in solution. The agents can be in soluble form and can be cross-linked to a surface such as a cell expressing an Fc receptor or an antibody or other binder that will bind to the agent. In this regard, reference is made, for example, to the artificial antigen-presenting cells (aAPCs) of U.S. Patent Application Publication Nos. 20040101519 and 20060034810, which are contemplated for use in activating and amplifying T cells in some aspects of the present disclosure.

[0352] In some aspects, T cells are combined with beads coated with a reagent, followed by separating the beads and the cells and culturing the cells. Prior to culturing, the beads coated with the agent and the cells may not be separated and may be cultured together. Cell stimulation can be induced by first concentrating the beads and cells by applying a force such as magnetic force to increase the ligation of cell surface markers.

[0353] For example, cell surface proteins can be ligated by enabling contact between T cells and paramagnetic beads (3×28 beads) to which anti-CD3 and anti-CD28 are attached. Cells (e.g., 10 4 ~4×10 8Individual T cells) and beads (e.g., anti-CD3 / CD28 MACSiBead particles with a recommended titer of 1:100) are combined in a buffer, e.g., PBS (without divalent cations, e.g., calcium and magnesium). One of ordinary skill in the art can readily understand that any cell concentration can be used. For example, the target cells can be present in very small amounts in the sample and may only account for 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells of interest. Therefore, any cell number is within the scope of the present disclosure. In order to ensure maximum contact between the cells and the particles, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the increase in cells). For example, a concentration of about 2 billion cells / mL may be used. More than 100 million cells / mL may be used. Concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / mL may be used. Concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / mL may be used. Concentrations of 125 million or 150 million cells / mL may be used. The use of high concentrations can result in an increase in cell yield, cell activation, and cell proliferation. In addition, the use of high cell concentrations can enable more effective capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells. Such cell populations can have therapeutic value and are expected to be obtainable in some embodiments. For example, the use of high concentrations of cells enables more effective selection of CD8+ T cells, which typically have weak CD28 expression.

[0354] The mixture can be cultured for any integer number of hours (about 3 hours) to about 14 days or both. The mixture can be cultured for 21 days. The beads and T cells are cultured together for about 8 days. The beads and T cells can be cultured together for 2 - 3 days. Several stimulation cycles may be required so that the culture time of the T cells can be 60 days or more. Conditions suitable for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which may contain factors essential for growth and survival, serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for cell growth well-known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium may be RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-vivo 20, an optimizer to which amino acids, sodium pyruvate, and vitamins are added and which is supplemented with no serum or an appropriate amount of serum (or plasma) or a defined group of hormones, and / or may contain a sufficient amount of cytokines to grow and amplify T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and not in cell cultures that are to be injected into a subject. The target cells are maintained under conditions essential for growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to different stimulation times can exhibit various characteristics. For example, typical peripheral blood mononuclear cell products of blood or apheresis blood components have more helper T cell populations (TH, CD4+) than cytotoxic or suppressor T cell populations (TC, CD8). T cell ex vivo amplification by stimulation of the CD3 and CD28 receptors produces a T cell population that is mainly composed of TH cells approximately 8 - 9 days before, and after about 8 - 9 days, the T cell population contains an increasing number of TC cell populations.Therefore, depending on the treatment objective, it may be advantageous to inject mainly a group of T cells including TH cells. Similarly, if an antigen-specific subgroup of TC cells is isolated, it may be beneficial to amplify the subgroup to a greater extent.

[0355] In addition to the CD4 and CD8 markers, other phenotypic markers are also significantly different but are fairly reproducible during cell proliferation. Therefore, due to such reproducibility, it becomes possible to customize T cell products activated for a specific purpose.

[0356] 5.4.6. CAR-T Cells Expressing Exogenously Introduced TGFβR and IL23R In some embodiments, the T cells according to this specification further express a chimeric receptor (also referred to herein as "TF23") that includes exogenously introduced TGFβR and IL23R.

[0357] More specifically, in some embodiments, CAR-T cells expressing exogenously introduced TGFβR and IL23R can be produced by introducing one or more nucleic acids encoding a polypeptide that includes both TGFβR and IL23R.

[0358] CAR, TGFβR, and IL23R can each be individually introduced into T cells as separate polypeptides. For example, a nucleic acid encoding the CAR according to this specification, a nucleic acid encoding TGFβR, and a nucleic acid encoding IL23R may each be introduced into T cells.

[0359] Alternatively, any two or all three of the three may be introduced together into a T cell as a single polypeptide by a single nucleic acid, which is cleaved when translated intracellularly. For example, introduce into a T cell a nucleic acid encoding a polypeptide comprising a CAR according to the present specification and a TGFβR linked via a self-cleaving peptide linker, and separately introduce into the T cell a nucleic acid encoding IL23R. Similarly, introduce into a T cell a nucleic acid encoding a polypeptide comprising a CAR according to the present specification and an IL23R linked via a self-cleaving peptide linker, and separately introduce into the T cell a nucleic acid encoding TGFβR. In some embodiments, a nucleic acid encoding a polypeptide comprising all three of CAR, TGFβR and IL23R linked to each other via a self-cleaving peptide linker may be introduced into a T cell. Above, the self-cleaving peptide linker is described in more detail. The 2A self-cleaving peptide may be selected from the group consisting of F2A, E2A, P2A, T2A or variants thereof. The self-cleaving peptide may be a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO:42.

[0360] Alternatively, the CAR-T cells according to the present specification can be produced from a polynucleotide comprising a plurality of regions, for example, a region encoding a CAR, a region encoding TGFβR and / or a region encoding IL23R. The different regions can be controlled by the same promoter. For example, in some embodiments, the present specification uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, the different regions are controlled by separate promoters.

[0361] In some specific embodiments, in the CAR-T cells according to this specification, a TF23 chimeric receptor is introduced exogenously, and the TF23 chimeric receptor includes a first extracellular domain containing the extracellular domain of TGFβR1, a first transmembrane domain containing the transmembrane domain of IL-12Rβ1, a first intracellular domain containing the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, a second extracellular domain containing the extracellular domain of TGFβR2, a second transmembrane domain containing the transmembrane domain of IL-23R, and a second intracellular domain containing the intracellular domain of IL-23R, as shown in Figure 7A. In some specific embodiments, the CAR-T cells according to this specification express an exogenously introduced polypeptide, and the polypeptide includes any one of the amino acid sequences of SEQ ID NO: 32 and 44-45. In other specific embodiments, a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 32, for example, a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 33, is introduced exogenously into the CAR-T cells according to this specification.

[0362] 5.5. Polynucleotide In some embodiments, the present disclosure provides a polynucleotide encoding an antibody of the present invention (e.g., a VHH domain antibody) that binds to claudin-6, and a fusion protein comprising an antibody that binds to claudin-6 described herein. The polynucleotide of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. The polynucleotide may be in the form of cDNA. The polynucleotide may be a synthetic polynucleotide.

[0363] In some embodiments, the present disclosure provides a polynucleotide encoding a Claudin-6 binding CAR according to this specification. The polynucleotides of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. The polynucleotide may be in the form of cDNA. The polynucleotide may be a synthetic polynucleotide.

[0364] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, antibodies or fragments, analogs, and / or derivatives of CARs that bind to Claudin-6 of the present disclosure. The present disclosure can provide a polynucleotide having a nucleotide sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to the polynucleotide encoding an antibody or CAR that binds to Claudin-6 of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide has identity to the reference nucleotide sequence, except that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of a plurality of nucleotides of the entire nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence can occur anywhere among single nucleotides in the reference sequence that are between or at the 5' or 3' terminal positions of the reference nucleotide sequence or within one or more contiguous groups within the reference sequence.

[0365] Polynucleotide variants can contain changes in the coding region, non-coding region, or both. Polynucleotide variants can cause silent substitutions, additions, or deletions, and may include changes that do not alter the properties or activities of the encoded polypeptide. Polynucleotide variants may include silent substitutions, which (due to the degeneracy of the genetic code) do not result in a change in the amino acid sequence of the polypeptide. Polynucleotide variants can be produced for various reasons, for example, to optimize the codon expression of a particular host (i.e., to change the codons of human mRNA to those preferred by a bacterial host such as E. coli). Polynucleotide variants may include at least one silent mutation in the non-coding or coding region of the sequence.

[0366] Polynucleotide variants can be produced to regulate or modify the expression (or expression level) of the encoded polypeptide. Polynucleotide variants can be produced to increase the expression of the encoded polypeptide. Polynucleotide variants can be produced to decrease the expression of the encoded polypeptide. Compared to the parental polynucleotide sequence, a polynucleotide variant may exhibit an increase in the expression of the encoded polypeptide. Compared to the parental polynucleotide sequence, a polynucleotide variant may exhibit a decrease in the expression of the encoded polypeptide.

[0367] Vectors comprising the nucleic acid molecules described herein are further provided. In one aspect, the nucleic acid molecules may be incorporated into recombinant expression vectors. The present disclosure provides recombinant expression vectors comprising any nucleic acid of the present disclosure. As used herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and wherein the vector, when contacted with a cell under conditions sufficient for the expression of the mRNA, protein, polypeptide, or peptide intracellularly, allows for expression via the host cell. The vectors described herein are not entirely naturally occurring; however, some vectors may be naturally occurring. The recombinant expression vectors described may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and which may contain natural, non-natural, or modified nucleotides. The recombinant expression vectors may comprise naturally occurring or non-naturally occurring nucleotide linkages, or both types of linkages. Non-naturally occurring or modified nucleotides or nucleotide linkages do not prevent transcription or replication of the vector.

[0368] The recombinant expression vectors of the present disclosure may be any suitable recombinant expression vectors and can be used to transform or transfect any suitable host. Suitable vectors are vectors designed to be used for performing proliferation and amplification or expression or both, for example, vectors including plasmids and viruses. The vectors may be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors, for example, λGT10, λGT11, λEMBL4, λNM1149 and λZapII (Stratagene) can be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, for example, a retroviral vector, for example, a γ-retroviral vector.

[0369] For example, recombinant expression vectors can be produced by standard recombinant DNA techniques described in the above Sambrook et al. and the above Ausubel et al. A circular or linear expression vector construct may be produced to include a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc.

[0370] The recombinant expression vector may include regulatory sequences, for example, transcription and translation start and termination codons, which are specific to the type of host (e.g., bacteria, plants, fungi or animals) into which the vector is introduced, determined by circumstances, taking into account whether the vector is DNA-based or RNA-based.

[0371] The recombinant expression vector may contain one or more marker genes, which allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts, providing prototrophic strains, etc. Suitable marker genes for the expression vector include, for example, the neomycin / G418 resistance gene, histidinol x resistance gene, histidinol resistance gene, tetracycline resistance gene, and ampicillin resista...

Claims

1. (i) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 7, respectively, (ii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO: 8, respectively, (iii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO: 9, respectively, (iv) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO: 10, respectively, or (v) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO: 11, respectively. A single-domain anti-claudin-6 antibody (sdAb) containing this antibody.

2. The anti-claudin-6 sdAb according to claim 1, wherein the CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof.

3. (i) CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 3, and CDR3 containing the amino acid sequence of SEQ ID NO: 5, or (ii) CDR1 containing the amino acid sequence of SEQ ID NO: 2, CDR2 containing the amino acid sequence of SEQ ID NO: 4, and CDR3 containing the amino acid sequence of SEQ ID NO: 6 The anti-claudin-6 sdAb according to claim 1 or 2, comprising:

4. The anti-claudin-6 sdAb according to claim 1, comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

5. The anti-claudin-6 sdAb according to claim 1, comprising or consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:

11.

6. A fusion protein comprising the anti-claudin-6 sdAb described in claim 1 and human IgG1Fc, wherein the fusion protein comprises any one amino acid sequence from SEQ ID NO: 21 to 25.

7. (a) an extracellular antigen-binding domain comprising the anti-claudin-6 sdAb described in claim 1, (b) Transmembrane domain and (c) Intracellular signaling domain and Chimeric antigen receptors (CARs), including those mentioned above.

8. The CAR according to claim 7, wherein the extracellular antigen-binding domain further comprises one or more further antigen-binding domains, and at least one of the further binding domains optionally binds to GPC3.

9. The aforementioned further antigen-binding domain, (i) HCDR1, HCDR2, and HCDR3, each having the amino acid sequences of HCDR1, HCDR2, and HCDR3 shown in VH containing the amino acid sequence of SEQ ID NO: 18, LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of LCDR1, LCDR2, and LCDR3 shown in VL containing the amino acid sequence of SEQ ID NO: 19; (ii) HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, HCDR3 containing the amino acid sequence of SEQ ID NO: 16, LCDR1 containing the amino acid sequence of SEQ ID NO: 13, LCDR2 containing the amino acid sequence of SEQ ID NO: 15, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17; (iii) A VH domain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of SEQ ID NO: 18, VL domains containing amino acid sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of SEQ ID NO: 19; (iv) A VH domain containing the amino acid sequence of SEQ ID NO: 18 and a VL domain containing the amino acid sequence of SEQ ID NO: 19; (v) An amino acid sequence having at least 80% identity with SEQ ID NO: 20; or (vi) Amino acid sequence of SEQ ID NO: 20 The CAR according to claim 8, including the following:

10. The antigen-binding domains are fused to each other via peptide linkers, where optionally, the length of the peptide linkers is approximately 50 amino acids or less. The CAR according to claim 8.

11. The transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, where optionally the transmembrane domain is derived from CD8α. The CAR according to claim 7.

12. The intracellular signaling domain includes the primary intracellular signaling domain of an immune effector cell, wherein the primary intracellular signaling domain is optionally derived from CD3ζ. The CAR according to claim 7.

13. The intracellular signaling domain further comprises a co-stimulatory signaling domain, wherein the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. Here, optionally, the co-stimulus signaling domain is derived from CD137. The CAR according to claim 12.

14. The domain further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, wherein the hinge domain is optionally derived from CD8α. The CAR according to claim 7.

15. The polypeptide further comprises a signal peptide located at its N-terminus, wherein the signal peptide is optionally derived from CD8α. The CAR according to claim 7.

16. A chimeric antigen receptor (CAR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26 and SEQ ID NO:

28.

17. A nucleic acid comprising a nucleic acid sequence encoding the anti-claudin-6 sdAb described in claim 1 or the fusion protein described in claim 6.

18. A nucleic acid comprising a nucleic acid sequence encoding the CAR described in claim 7.

19. The nucleic acid according to claim 18, wherein the nucleic acid further comprises a nucleic acid sequence encoding a chimeric receptor, the chimeric receptor comprises TGFβR and IL23R, and optionally, the nucleic acid encoding the chimeric receptor comprises the nucleic acid sequence of SEQ ID NO:

33.

20. A chimeric receptor containing one of the amino acid sequences from SEQ ID NO: 32 and 44-45.

21. A vector comprising the nucleic acid described in claim 17.

22. Engineered immunoeffector cells comprising the CAR according to claim 7, the chimeric receptor according to claim 20, the nucleic acid according to claim 17, or the vector according to claim 21.

23. The manipulated immune effector cells according to claim 22, which are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, or a combination thereof.

24. A method for producing engineered immunoeffector cells, comprising introducing the vector according to claim 21 into cells.

25. A pharmaceutical composition for use in a method of treating a disease or disorder in a subject, comprising anti-claudin-6 sdAb according to claim 1, nucleic acid according to claim 17, vector according to claim 21, engineered immune effector cells according to claim 22, and a pharmaceutically acceptable excipient.

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