Tight junction protein 6 binding moieties and uses thereof

Abstract

The present disclosure provides an anti-tight junction protein 6 antibody (e.g., a VHH domain antibody), and a chimeric antigen receptor (CAR) that binds tight junction protein 6 comprising: the anti-tight junction protein 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 in cancer immunotherapy.

Description

[0001] Cross-references

[0002] This application claims the priority benefit of international patent application No. PCT / CN2022 / 109077 filed on July 29, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application incorporates by reference the sequence listing submitted with this application, which is in XML file format, titled "IEC230078PCT-seql.xml", created on July 26, 2023, and is 68,969 bytes in size. 1. Technical Field

[0005] The present disclosure relates to the field of antibodies (eg, domain antibodies), chimeric antigen receptors, and engineered immune cells that target Claudin 6, and methods of using the same. 2. Background technology

[0006] Claudin (CLDN) is an important tight junction protein that is mainly expressed in endothelial cells or epithelial cells in a tissue-specific manner (see Int J Mol Sci, 14; 22 (24): 13416 (2021)). As a member of the CLDN family, tight junction protein 6 (CLDN6) has been found to play a key role in the formation of barriers, especially lung epithelial barriers and epidermal permeability barriers (EPB). There is evidence that changes in CLDN6 expression are associated with the development of various cancers, and the malignant phenotypes of tumors affected by CLDN6 include proliferation and apoptosis, migration and invasion, and drug resistance, which are all regulated by key signaling pathways mediated by CLDN6. CLDN6 is an oncofetal target that has been shown to be highly expressed in certain solid tumors but not in normal tissues (see Histopathology, 61 (6): 1043-56 (2021); Sci Transl Med., 13 (579): eabb6282 (2021)). The same expression profile was shown in another oncofetal target, GPC3. There is a need in the art for new anti-claudin 6 binding proteins and immunotherapies, such as CAR-T therapies targeting claudin 6. 3. Summary of the invention

[0007] In one aspect, the present invention provides an anti-claudin 6 (CLDN6) single domain antibody (sdAb), comprising (i) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:7; (ii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:8; (iii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:9; (iv) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:10; or (v) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as 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.

[0008] In one aspect, the present invention provides an anti-claudin 6 single domain antibody (sdAb), comprising: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5; or (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0009] In some embodiments, the anti-Claudin 6 sdAb provided herein further comprises one or more FR regions as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and / or SEQ ID NO:11.

[0010] In some embodiments, provided herein is an anti-claudin 6 sdAb 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. In some embodiments, provided herein is an anti-claudin 6 sdAb comprising or consisting 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: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0011] In some embodiments, the anti-Claudin 6 sdAb is a camelid sdAb. In some embodiments, the anti-Claudin 6 sdAb is a humanized sdAb.

[0012] In some embodiments, the anti-Claudin 6 sdAb is genetically fused or chemically conjugated to the agent.

[0013] In another aspect, the present invention provides a fusion protein comprising an anti-claudin 6 sdAb provided herein and an Fc region (eg, human IgG1 Fc). In some embodiments, 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 NO: 21-25.

[0014] In another aspect, provided herein is a chimeric antigen receptor (CAR) comprising: an extracellular antigen binding domain comprising the anti-claudin 6 sdAb provided herein; a transmembrane domain; and an intracellular signaling domain.

[0015] 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 domains comprise: HCDR1, HCDR2, and HCDR3 having the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, as shown in VH comprising the amino acid sequence of SEQ ID NO: 18; and LCDR1, LCDR2, and LCDR3 having the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, as shown in VL comprising the amino acid sequence of SEQ ID NO: 19.

[0016] In some embodiments, the additional antigen binding domain comprises: HCDR1, which HCDR1 comprises the amino acid sequence of SEQ ID NO:12; HCDR2, which HCDR2 comprises the amino acid sequence of SEQ ID NO:14; HCDR3, which HCDR3 comprises the amino acid sequence of SEQ ID NO:16; LCDR1, which LCDR1 comprises the amino acid sequence of SEQ ID NO:13; LCDR2, which LCDR2 comprises the amino acid sequence of SEQ ID NO:15; and LCDR3, which LCDR3 comprises the amino acid sequence of SEQ ID NO:17.

[0017] In some embodiments, the additional antigen binding domain comprises a VH domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 18, and a VL domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the additional antigen binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 18, and a VL domain comprising the amino acid sequence of SEQ ID NO: 19.

[0018] 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 that is at least 80% identical to SEQ ID NO:20.

[0019] In some embodiments, the antigen binding domains are fused to each other via a peptide linker.

[0020] In some embodiments, the peptide linker is no more than about 50 amino acids long.

[0021] 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.

[0022] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta.

[0023] In some embodiments, the intracellular signaling domain further 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 a ligand of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and a combination thereof. In some embodiments, the co-stimulatory signaling domain is derived from CD137.

[0024] In some embodiments, the CAR provided herein further comprises a hinge domain 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 CD8 α.

[0025] In some embodiments, the CAR provided herein further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD8α.

[0026] In some embodiments, provided herein is a chimeric antigen receptor (CAR) comprising: (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 to the sequences of SEQ ID NO: 26 and SEQ ID NO: 28.

[0027] In other aspects, provided herein is a nucleic acid encoding the anti-Claudin 6 sdAb, fusion protein or CAR provided herein, or a fragment thereof.

[0028] In other aspects, the nucleic acids provided herein further comprise a sequence encoding a chimeric receptor. In some embodiments, the nucleic acid encoding the chimeric receptor comprises a nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid encoding the chimeric receptor encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 32 and 44-45.

[0029] In other aspects, provided herein is a vector comprising a nucleic acid provided herein.

[0030] In other aspects, there is provided herein a kind of engineered immune effector cell, it includes CAR provided herein, nucleic acid and / or carrier.In certain embodiments, engineered immune effector cell is T cell, NK cell, peripheral blood mononuclear cell (PBMC), hematopoietic stem cell, pluripotent stem cell, embryonic stem cell or their combination.

[0031] In other aspects, provided herein is a pharmaceutical composition comprising the anti-claudin 6 sdAb provided herein, a nucleic acid, a vector or an engineered immune effector cell, and a pharmaceutically acceptable excipient.

[0032] In other aspects, there is provided herein a method for treating a disease or condition of a subject, the method comprising administering to the subject an effective amount of an anti-tight junction protein 6 sdAb, an engineered immune effector cell or a pharmaceutical composition provided herein. In some embodiments, the disease or condition is a tight junction protein 6-related disease or condition, and / or a GPC3-related disease or condition. In some embodiments, the disease or condition is a cancer, such as a solid tumor cancer. In some embodiments, the disease or condition is selected from the group consisting of: germ cell tumor (GCT), ovarian cancer (OC), non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), endometrioid carcinoma (EC) and AFP+ gastric cancer (AFP+GC).

[0033] In other aspects, the present disclosure also provides a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a combination of a CLDN6 antagonist and a GPC3 antagonist, or an antagonist of CLDN6 and GPC3. The CLDN6 antagonist, the GPC3 antagonist and / or the antagonist of CLDN6 and GPC3 may be of various types, for example, the antagonist may be selected from engineered receptors, engineered immune cells, antibodies, antibody-drug conjugates (ADCs), aptamers, and small RNAs.

[0034] On the one hand, the CLDN6 antagonist is an engineered immune cell comprising an engineered receptor specifically targeting CLDN6, the engineered receptor comprising: an extracellular antigen binding domain comprising at least one anti-CLDN6 binding portion (e.g., an anti-CLDN6sdAb provided herein), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the GPC3 antagonist is an engineered immune cell comprising an engineered receptor specifically targeting GPC3, the engineered receptor comprising: an extracellular antigen binding domain comprising at least one anti-GPC3 binding portion, a transmembrane domain, and an intracellular signaling domain. In some embodiments, an engineered receptor specifically targeting CLDN6 (e.g., a CLDN6CAR provided herein) and an engineered receptor specifically targeting GPC3 are expressed in different engineered immune cells. Accordingly, the combination of a CLDN6 antagonist and a GPC3 antagonist is a combination of a first group of engineered immune cells comprising an engineered receptor specifically targeting CLDN6 as described herein and a second group of engineered immune cells comprising an engineered receptor specifically targeting GPC3.

[0035] In some embodiments, the antagonist of CLDN6 and GPC3 is an engineered immune cell comprising 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 antagonist of CLDN6 and GPC3 is an engineered immune cell comprising a first engineered receptor that specifically targets CLDN6 and a second engineered receptor that specifically targets GPC3, wherein (1) the first engineered receptor that specifically targets CLDN6 comprises: a first extracellular antigen binding domain comprising at least one anti-CLDN6 binding portion, a first transmembrane domain, and a first intracellular signaling domain, and (2) the second engineered receptor that specifically targets GPC3 comprises: a second extracellular antigen binding domain comprising at least one anti-GPC3 binding portion, a second transmembrane domain, and a second intracellular signaling domain.

[0036] In some embodiments, the antagonist of CLDN6 and GPC3 is an engineered immune cell comprising a first CAR targeting CLDN6 and a second CAR targeting GPC3, wherein (1) the first CAR comprises a first extracellular antigen binding domain comprising at least one anti-CLDN6 binding portion (e.g., an anti-CLDN6 sdAb provided herein), a first transmembrane domain, and a first intracellular signaling domain, (2) the second CAR comprises a second extracellular antigen binding domain comprising at least one anti-GPC3 binding portion, a second transmembrane domain, and a second intracellular signaling domain; and wherein the first CAR is operably linked to the second CAR, the first CAR is located at the N-terminus or C-terminus of the second CAR targeting GPC3, optionally the first CAR is operably linked to the second CAR via a cleavable peptide, or the first CAR and the second CAR are not linked due to cleavage of the cleavable peptide.

[0037] In some embodiments, the antagonist of CLDN6 and GPC3 is an engineered immune cell comprising an engineered receptor that co-targets CLDN6 and GPC3, the engineered receptor comprising: an extracellular antigen binding domain comprising at least one anti-CLDN6 binding portion (e.g., an anti-CLDN6 sdAb provided herein) and at least one anti-GPC3 binding portion, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the antagonist of CLDN6 and GPC3 is an engineered immune cell comprising a CAR that co-targets CLDN6 and GPC3 ("CLDN6×GPC3 bispecific CAR"), wherein the CLDN6×GPC3-specific CAR comprises: (1) an extracellular antigen binding domain comprising at least one anti-CLDN6 binding portion and at least one anti-GPC3 binding portion, (2) a transmembrane domain, and (3) an intracellular signaling domain; and wherein the anti-CLDN6 binding portion is located at the N-terminus or C-terminus of the anti-GPC3 binding portion, optionally the anti-CLDN6 binding portion is operably linked to the anti-GPC3 binding portion via a peptide linker such as a GS linker, e.g., (G4S)3.

[0038] In some embodiments, the disease or condition is a claudin 6-related disease or condition, and / or a GPC3-related disease or condition. In some embodiments, the disease or condition is a cancer, such as a solid tumor cancer. In some embodiments, the disease or condition is selected from the group consisting of germ cell tumors (GCT), ovarian cancer (OC), non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), endometrioid carcinoma (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. 4. Description of the drawings

[0040] Figures 1A-1E Binding of the chimeric antibody to ES-2.huCLDN.Luc cells and OV90 cells is shown.

[0041] Figure 2A-2E Binding of the humanized chimeric antibody to ES-2.huCLDN.Luc cells and OV90 cells is shown.

[0042] Figures 3A-3C The in vitro cytotoxicity of CAR-T against different CLDN families is shown. "UnT" indicates T cells not transduced with CAR. "CD19CAR-T" is used as an irrelevant control CAR-T.

[0043] Figures 4A-4DIFN-γ release by CAR-T against different CLDN families is shown. "UnT" indicates T cells not transduced with CAR. "CD19CAR-T" was used as an irrelevant control CAR-T.

[0044] Figures 5A-5C The in vivo efficacy of CLDN6CAR-T cells in OV90.Luc xenograft mice is shown. "UnT" indicates T cells not transduced with CAR.

[0045] Figure 6 In vitro cytotoxicity of CAR-T cells co-cultured with tumor cells with different GPC3 and CLDN6 expression levels is shown. "UnT" indicates T cells not transduced with CAR.

[0046] Fig. 7A Exogenously introduced TGFβR and IL23R constructs are illustrated. Figure 7B The expression of GPC3 and CLDN6 in subcutaneous tumor sections of OV90.Luc cells is shown.

[0047] Figures 8A-8B The in vivo efficacy of CAR-T cells in OV90.Luc xenograft mice is shown (0.2M CAR+ / mouse). "UnT" indicates T cells not transduced with CAR. 5. Specific implementation methods

[0048] The present disclosure is based, in part, on novel antibodies that bind Claudin 6, chimeric antigen receptors that bind Claudin 6 and / or GPC3, or engineered cells comprising them and / or co-expressing chimeric receptors, and their improved properties.

[0049] 5.1. Definitions

[0050] The techniques and procedures described or referenced herein include those generally well understood and / or commonly employed by those skilled in the art using routine methods, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al., eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies :Methods and Protocols (Albitar ed. 2010); and AntibodyEngineeringVolume 1 and Volume 2 (Kontermann and Dübel eds., 2nd edition 2010). Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following description of the terms will apply, and whenever appropriate, terms used in the singular will also include the plural, and vice versa. In the event that any description of the stated terms conflicts with any document incorporated herein by reference, the description of the terms stated below shall prevail.

[0051] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and in the broadest sense, and specifically encompass, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with 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 fragments thereof (e.g., domain antibodies), as described below. The antibody may be a human antibody, a humanized antibody, a chimeric antibody, and / or an affinity matured antibody, as well as antibodies from other species (e.g., mouse, rabbit, llama, etc.). The term "antibody" is intended to include the polypeptide products of B cells in the class of immunoglobulin polypeptides, which are capable of binding to a specific molecular antigen and are composed of two pairs of identical polypeptide chains, wherein 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 includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain includes a constant region. See, e.g. Antibodey Engineering (Borrebaeck ed., 2nd ed. 1995) and Kuby, Immunology(3rd edition 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, antibodies including those from camelid species (e.g., llamas or alpacas) or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the above, which refer to a portion of an antibody heavy chain or light chain polypeptide that retains 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 Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. Specifically, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules containing antigen binding sites that bind to antigens (e.g., one or more CDRs of antibodies). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd edition 1990). The antibodies provided herein can be any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibodies can be agonistic antibodies or antagonistic antibodies. The antibodies can be neither agonistic nor antagonistic.

[0052] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. A target antigen can be a polypeptide. An antigen can be associated with a cell, for example, present on or in a cell.

[0053] A "complete" antibody is an antibody that comprises an antigen binding site as well as CL and at least heavy chain constant regions CH1, CH2 and CH3. The constant region may include a human constant region or an amino acid sequence variant thereof. A complete antibody may have one or more effector functions.

[0054] "Single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment comprising VH and VL antibody domains connected to a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0055] The term "heavy chain only antibody" or "HCAb" refers to a functional antibody that comprises a heavy chain but lacks the light chain typically found in a 4-chain antibody. For example, camelids (such as camels, llamas or alpacas) are known to produce HCAbs.

[0056] As used herein, "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 tight junction protein 6). Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies that naturally lack light chains, such as antibodies from camelid species (e.g., llamas), single domain antibodies derived from conventional 4-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, mice, humans, camels, llamas, goats, rabbits, and cattle. For example, as described herein, single domain antibodies may be derived from camelid species, such as antibodies produced in camels, llamas, dromedaries, alpacas, and guanacos. Other species except camelids may produce heavy chain antibodies that naturally lack light chains; VHHs derived from such other species are within the scope of the present disclosure. In some embodiments, the single domain antibodies (e.g., VHH) provided herein have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The single domain antibody can be genetically fused or chemically conjugated with another molecule (e.g., medicament) as described herein. The single domain antibody may be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0057] The term "binding" refers to the interaction between molecules, including, for example, the formation of a complex. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the combination of two or more molecules that are bound together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interaction between a single antigen binding site on an antibody and a single epitope of a target molecule (such as an antigen) is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k) of a binding molecule (e.g., an antibody) from a monovalent antigen is the affinity of the antibody or functional fragment for that epitope. off ) and the association rate (k on ) ratio (k off / k on ) is the dissociation constant K D , which is inversely proportional to affinity. K D The lower the value, the higher the affinity of the antibody. D The value varies for different complexes of antibody and antigen and depends on the k on and k off The dissociation constant K of the antibodies provided herein D Can use any method provided herein or any other method well known to those skilled in the art to determine.The affinity at a binding site does not always reflect the true intensity of interaction between antibody and antigen.When a composite antigen (such as a multivalent antigen) containing multiple repeated antigenic determinants contacts with an antibody containing multiple binding sites, the interaction of antibody and antigen at one site will increase the possibility of reaction at a second site.The intensity of this multiple interaction between multivalent antibody and antigen is called avidity.

[0058] With respect to the binding molecules described herein, terms such as "binding," "specifically binding," and similar terms are also used interchangeably herein and refer to a binding molecule that specifically binds to an antigen binding domain of an antigen (such as a polypeptide). A binding molecule or antigen binding domain that binds or specifically binds to an antigen can be detected, for example, by immunoassays, Or other techniques known to those skilled in the art. A binding molecule or antigen binding domain can bind or specifically bind to an antigen when it binds to the antigen with a higher affinity than it binds to any cross-reactive 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 twice the background signal or noise, and can be more than 10 times the background. See, e.g. Fundamental Immunology332-36 (Paul ed., 2nd ed. 1989) for a discussion of binding specificity. The extent of binding of a binding molecule or antigen binding domain to a "non-target" protein can be about 10% of the binding of the binding molecule or antigen binding domain to its specific target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA. Binding molecules or antigen binding domains that bind to an antigen include binding molecules or antigen binding domains that are capable of binding to an antigen with sufficient affinity so that the binding molecule can be used, for example, as a therapeutic and / or diagnostic agent targeting an antigen. A binding molecule or antigen binding domain that binds an antigen can have a dissociation constant (K) of less than or equal to 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. D ). The binding molecule or antigen binding domain binds to an epitope that may be conserved in antigens from different species.

[0059] Binding molecules or antigen binding domains may comprise "chimeric" sequences, wherein a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-55). Chimeric sequences may include humanized sequences.

[0060] Binding molecules or antigen binding domains can include portions of "humanized" forms of non-human (e.g., camelids, mice, non-human primates) antibodies, which include sequences from human immunoglobulins (e.g., receptor antibodies), wherein natural CDR residues are replaced by residues from non-human species (e.g., donor antibodies) such as camelids, mice, rats, rabbits or non-human primates with desired specificity, affinity and ability. One or more FR region residues of human immunoglobulin sequences can be replaced by corresponding non-human residues. In addition, humanized antibodies can be included in residues not found in receptor antibodies or donor antibodies. These modifications are made to further improve antibody performance. Humanized antibody heavy or light chains can include substantially all at least one or more variable regions, wherein all or substantially all CDRs correspond to the CDRs of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences. Humanized antibodies will include at least a portion of an immunoglobulin constant region (Fc), typically 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.

[0061] A binding molecule or antigen binding domain may comprise a portion of a "fully human antibody" or "human antibody", wherein these terms are used interchangeably herein and refer to antibodies comprising human variable regions and, for example, human constant regions. A binding molecule may comprise an antibody sequence. The term refers to an antibody that may comprise variable and constant regions of human origin. A "fully human" antibody may also encompass antibodies that bind to a polypeptide and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies having variable and constant regions that correspond to human germline immunoglobulin sequences, as described by Kabat et al. (see Kabat et al. (1991)). Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human being and / or has been produced using any technique for making human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using various 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 to prepare human monoclonal antibodies are described in the following literature: 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 prepared by administering an antigen to a transgenic animal (e.g., mouse) that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6 (5): 561-66 (1995); Brüggemann and Taussing, Curr. Opin. Biotechnol. 8 (4): 455-58 (1997); and about XENOMOUSE TM For human antibodies generated by human B cell hybridoma technology, see also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006).

[0062] Binding molecules or antigen binding domains may comprise portions of "recombinant human antibodies", wherein the phrase includes human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, antibodies isolated from animals (e.g., mice or cattle) that are transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20:6287-6295 (1992)) or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire in vivo.

[0063] Binding molecules or antigen binding domains can include a portion of a "monoclonal antibody", wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, for example, except for possible naturally occurring mutations or well-known post-translational modifications (such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation) that may be present in small amounts, the individual antibodies constituting the population are identical, and each monoclonal antibody will generally recognize a single epitope on the antigen. As used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for preparing an antibody. For example, the monoclonal antibodies that can be used for the present disclosure can be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be prepared using recombinant DNA methods in bacteria or eukaryotic animals or plant cells (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art. See, e.g. Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0064] A typical 4-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 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds according to the H chain isotype. Each H chain and L chain also have 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 chain and the γ chain, and four CH domains for the μ isotype and the ε isotype. Each L chain has a variable domain (VL) at the N-terminus and a constant domain (CL) at the other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are considered to form an interface between the light chain and the heavy chain variable domains. The pairing of VH and VL together forms a single antigen binding site. For the structure and properties of different classes of antibodies, see e.g. Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994) and Immunobiology (Janeway et al., 5th edition 2001).

[0065] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgG generally comprises two Fab regions, each of which is located on one of the two arms of a Y-shaped IgG structure. Each Fab region generally consists of a variable region and a constant region of each of a heavy chain and a light chain. More specifically, the variable region and constant region of the heavy chain in the Fab region are the VH region and the CH1 region, and the variable region and constant region of the light chain in the Fab region are the VL region and the CL region. The VH, CH1, VL and CL in the Fab region can be arranged in various ways to impart antigen binding ability according to the present disclosure. For example, the VH region and the CH1 region can be on one polypeptide, and the VL region and the CL region can be on a separate polypeptide, similar to the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following section.

[0066] The term "variable region", "variable domain", "V region" or "V domain" refers to a portion of the light or heavy chain of an antibody, which is usually located at the amino terminus of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain can be called "VH". The variable region of the light chain can be called "VL". The term "variable" refers to the fact that some segments of the variable region in an antibody are very different in sequence. The V region mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, the variability is not evenly distributed within the 110 amino acids of the variable region. Instead, the V region consists of segments of about 15-30 amino acids called framework regions (FRs) that are less variable (e.g., relatively unchanged), separated by shorter regions of greater variation (e.g., extreme variation) called "hypervariable regions" that are each about 9-12 amino acids long. The variable regions of the heavy and light chains each contain four FRs, primarily in a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th edition, 1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region between different antibodies is very different in sequence. In a specific embodiment, the variable region is a human variable region.

[0067] The term "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system for heavy chain variable regions or light chain variable regions used in the above-mentioned Kabat et al. compilation of antibodies. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. 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, etc. according to Kabat). The Kabat numbering of the residues of a given antibody can be determined by comparing the antibody sequence with the homology region of the "standard" Kabat numbering sequence. When referring to the 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., Kabat et al., above) is generally used. When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" (e.g., the EU index reported by Kabat et al., supra) is generally used. The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0068] When used to refer to antibodies, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino terminal portion includes a variable region of about 120 to 130 or more amino acids, and the carboxyl terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isotypes) known as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Different heavy chains are of different sizes: 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 produce antibodies of five well-known categories (e.g., isotypes), IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG, i.e., IgG1, IgG2, IgG3, and IgG4.

[0069] When used to refer to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) or lambda (λ).

[0070] As used herein, the terms "hypervariable region", "HVR", "complementarity determining region" and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the VH β-fold framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the VL β-fold 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. Therefore, CDRs are variable region sequences interspersed within the framework region sequences.

[0071] CDR regions are well known to those skilled in the art and are defined by a well-known numbering system. For example, Kabat complementary determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al., above; Nick Deschacht et al., J Immunol 2010; 184: 5696-5704). Chothia refers to the position of the structural loop instead (see, for example, Chothia and Lesk, J. Mol. Biol. 196: 901-17 (1987)). When numbering using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see e.g. Antibody Engineering Volume 2 (Kontermann and Dübel eds., 2nd edition 2010)). The "contact" hypervariable regions are based on analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System. (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77(2003)). IMGT is a comprehensive information system dedicated to the immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates. In this article, CDR refers to the amino acid sequence and position within the light chain or heavy chain. Since the "position" of CDR within the structure of the immunoglobulin variable domain is conserved between species and exists in structures called loops, it is easy to identify CDR and framework residues by using a numbering system that aligns the variable domain sequences according to structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into an acceptor framework, usually from a human antibody. Honegger and Plückthun, J. Mol. Biol. 309:657-70 (2001) developed another numbering system (AHon). The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT unique 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 each of these hypervariable regions or CDRs are exemplified in Table 1 below.

[0072] Table 1. Exemplary CDRs according to various numbering systems

[0073] The boundary of a given CDR can be different according to the scheme for identification.Therefore, unless otherwise stated, the term "CDR" and "complementarity determining region" of a given antibody or its district (such as a variable region) and each CDR (such as CDR-H1, CDR-H2) of an antibody or its district should be understood to encompass the complementary determining region defined by any known scheme as described above.In some cases, the scheme for identifying one or more specific CDRs is specified, such as the CDR defined by IMGT, Kabat, Chothia or Contact methods.In other cases, the specific amino acid sequence of CDR is given.It should be noted that the CDR district can also be defined by the combination of various numbering systems, such as the combination of Kabat and Chothia numbering systems, the combination of Kabat and AbM numbering systems or the combination of Kabat and IMGT numbering systems.Therefore, terms such as "CDR1 as set forth in a specific VH" include any CDR1 defined by the above-mentioned exemplary CDR numbering system, but are not limited thereto.Once a variable region (such as, VH or VL) is given, it will be understood by those skilled in the art that the CDR in the district can be defined by different numbering systems or their combinations.

[0074] The hypervariable regions may include the following "extended hypervariable regions": 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.

[0075] The term "constant region" or "constant domain" refers to the carboxyl terminal portion of the light and heavy chains that is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as interaction with Fc receptors. The term refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin, the variable region, which 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.

[0076] The term "framework" or "FR" refers to those variable region residues located on both sides of the CDR. FR residues are present in, for example, chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0077] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may be different, a human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at a Cys226 or Pro230 position to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region may be removed, for example, during the production or purification of the antibody, or by recombinant engineering of nucleic acids encoding the heavy chain of the antibody. Therefore, the composition of a complete antibody may include an antibody group with all K447 residues removed, an antibody group without the K447 residue removed, and an antibody group with a mixture of antibodies containing and not containing the K447 residue. A "functional Fc region" has an "effector function" 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), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain), and can be evaluated using various assays known to those skilled in the art. A "variant Fc region" comprises an amino acid sequence that is different from a native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the Fc region of the native sequence Fc region or a parent polypeptide, for example, about one to about ten amino acid substitutions in the Fc region of the native sequence Fc region or a parent polypeptide, or about one to about five amino acid substitutions. The variant Fc region herein may have at least about 80% homology with the Fc region of the native sequence Fc region and / or a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0078] 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 can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be a continuous amino acid of a polypeptide (a "linear" epitope), or an epitope can comprise amino acids from two or more discontinuous regions of a polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). It will be appreciated by those skilled in the art that, in general, a linear epitope can be dependent on or independent of a secondary, tertiary, or quaternary structure. For example, a binding molecule can bind to a group of amino acids, whether or not these amino acids are folded in a native three-dimensional protein structure. A binding molecule may require the amino acid residues constituting the epitope to exhibit a specific conformation (e.g., bending, twisting, turning, or folding) to recognize and bind to the epitope.

[0079] "Percent (%) sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. Publicly available calculator software such as BLAST, BLAST-2, ALIGN or MEGALIGN can be used in a variety of ways within the skill of the art. TM (DNASTAR) software is used to achieve alignment to determine the percentage of amino acid sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

[0080] The term "specificity" refers to the selective recognition of antigen-specific epitopes by antigen-binding proteins (such as CAR or antibodies). For example, natural antibodies are monospecific. As used herein, the term "multispecific" means that an antigen-binding protein (such as CAR or antibody) has two or more antigen binding sites, at least two of which bind to different antigens. As used herein, "bispecific" means that an antigen-binding protein (such as CAR or antibody) has two different antigen-binding specificities. As used herein, the term "monospecific" CAR means an antigen-binding protein (such as CAR or antibody) with one or more binding sites, each binding site binding to the same antigen.

[0081] As used herein, the term "valence" means that there are a specified number of binding sites in an antigen binding protein (such as a CAR or an antibody). For example, a natural antibody or a full-length antibody has two binding sites and is divalent. Thus, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" respectively represent that there are two binding sites, three binding sites, four binding sites, five binding sites and six binding sites in an antigen binding protein (such as a CAR or an antibody).

[0082] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to transplant one or more antigen-specifics to 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". CAR may include an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain of T cells and / or other receptors that are specific to one or more antigens (such as tumor antigens). "CAR-T cells" refer to T cells expressing CAR.

[0083] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes, for example, polypeptides containing one or more amino acid analogs, including but not limited to non-natural amino acids, as well as 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 chain or as two or more related chains.

[0084] 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 by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, usually single-stranded, synthetic polynucleotide, which is usually (but not necessarily) less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells that produce the binding molecules of the present disclosure may include parent hybridoma cells, as well as bacteria and eukaryotic host cells into which nucleic acids encoding antibodies have been introduced. Unless otherwise indicated, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is called the transcription direction; the sequence region on the DNA chain from 5' to the 5' end of the RNA transcript that has the same sequence as the RNA transcript is called the "upstream sequence"; the sequence region on the DNA chain from 3' to the 3' end of the RNA transcript that has the same sequence as the RNA transcript is called the "downstream sequence".

[0085] "Isolated nucleic acid" is such a nucleic acid (e.g., RNA, DNA, or mixed nucleic acid): it is substantially separated from other genomic DNA sequences and proteins or complexes such as ribosomes and polymerases that accompany natural sequences. "Isolated" nucleic acid molecules are nucleic acid molecules separated from other nucleic acid molecules present in the natural source of nucleic acid molecules. In addition, "isolated" nucleic acid molecules, such as cDNA molecules, when produced by recombinant technology, may be substantially free of other cell materials or culture medium, or when chemically synthesized, substantially free of chemical precursors or other chemicals. One or more nucleic acid molecules encoding antibodies described herein may be separated or purified. The term includes nucleic acid sequences removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized by heterologous systems. Substantially pure molecules may include isolated forms of the molecule. Specifically, the "isolated" nucleic acid molecules encoding CAR or antibodies described herein are nucleic acid molecules identified and separated from at least one contaminant nucleic acid molecule, which are usually associated with at least one contaminant nucleic acid molecule in the environment in which they are produced.

[0086] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that the nucleotide sequence encoding the protein may contain one or more introns in certain forms.

[0087] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0088] As used herein, the term "operably linked" and similar phrases (e.g., genetically fused), when used to refer to nucleic acids or amino acids, refer to the operable connection of nucleic acid sequences or amino acid sequences, respectively, which are in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the accurate production of nucleic acid molecules (e.g., RNA). Operably linked nucleic acid elements can result in the transcription of the open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide is one in which the functional domains are placed at an appropriate distance from each other to impart the intended function of each domain.

[0089] The term "vector" refers to a substance for carrying or including a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., antibody) as described herein, to introduce the nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include a selection sequence or a marker that may be stably integrated into a host cell chromosome. In addition, the vector may include one or more selective marker genes and appropriate expression control sequences. The selective marker gene that may be included may, for example, provide resistance to antibiotics or toxins, supplement nutritional deficiencies, or supply key nutrients that are not present in the culture medium. The expression control sequence may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., antibody heavy chain and light chain or antibody VH and VL), two nucleic acid molecules may be inserted, for example, in a single expression vector or a separate expression vector. For single vector expression, the encoding nucleic acid may be operably linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art may be used to confirm that nucleic acid molecules are introduced into a host cell. These methods include, for example, nucleic acid analysis, such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of introduced nucleic acid sequences or their corresponding gene products. It will be understood by those skilled in the art that nucleic acid molecules are expressed in an amount sufficient to produce the desired product, and it will be further understood that methods well known in the art can be used to optimize expression levels to obtain sufficient expression.

[0090] As used herein, the term "host" refers to an animal, such as a mammal (eg, a human).

[0091] As used herein, the term "host cell" refers to a specific target cell that can be transfected with a nucleic acid molecule, as well as the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or the integration of the nucleic acid molecule into the host cell genome.

[0092] As used herein, the term "autologous" is intended to refer to any material derived from the same individual, wherein the material is subsequently reintroduced into the individual.

[0093] "Allogeneic" refers to a transplant that originates from a different individual of the same species.

[0094] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous 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 cell includes the primary target cell and its progeny.

[0095] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or in United States Pharmacopeia , European Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0096] "Excipient" refers to pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, solvents or encapsulating materials. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, adhesives, buffers, carriers, coatings, coloring agents, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, wetting agents, lubricants, spices, preservatives, propellants, release agents, sterilants, sweeteners, solubilizers, wetting agents and mixtures thereof. The term "excipient" can also refer to diluents, adjuvants (e.g., Freund's adjuvant (complete or incomplete)) or vehicles.

[0097] 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; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; 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 dextrins; 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 TM , polyethylene glycol (PEG) and PLURONICS TM Other examples of pharmaceutically acceptable excipients are given in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th edition, 1990).

[0098] Each component can be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th edition; Rowe et al., eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd edition; Ash and Ash, eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd edition; Gibson, ed.; CRC Press LLC: Boca Raton, FL, 2009. A pharmaceutically acceptable excipient can be nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. A pharmaceutically acceptable excipient can be an aqueous pH buffered solution.

[0099] Excipients can be sterile liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary excipient. Saline solution and aqueous glucose solution and glycerol solution can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum powder, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier or pH buffer. The composition can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. Oral compositions, including preparations, can include standard excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0100] Compositions including a pharmaceutical compound may contain, for example, a binding molecule (eg, an antibody) in isolated or purified form and a suitable amount of an excipient.

[0101] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an antibody or therapeutic molecule comprising an agent and an antibody or pharmaceutical composition provided herein sufficient to produce a desired result.

[0102] The terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate or a primate (e.g., a human). A subject can be a human. A subject can be a mammal, such as a human, diagnosed with a disease or condition. Alternatively, a subject is a mammal, such as a human, at risk of developing a disease or condition.

[0103] "Administering" refers to the act of injecting or otherwise physically delivering a substance present outside the body into a patient's body, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0104] As used herein, the term "treat" refers to reducing or ameliorating the progression, severity and / or duration of a disease or condition caused by the administration of one or more therapies. Treatment can be determined by assessing whether one or more symptoms associated with the underlying condition have been reduced, alleviated and / or mitigated, such that an improvement in the patient is observed, although the patient may still be suffering from the underlying condition. The term "treat" includes controlling and ameliorating a disease. The term "control" refers to the beneficial effects that a subject obtains from a therapy that does not necessarily result in a cure of the disease.

[0105] The term "antagonist" refers to a substance that interferes with or inhibits the physiological effects of a target antigen or a signal transduction pathway mediated by a target antigen. Various types of antagonists are known in the art, including but 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 RNAs, and chemical compound inhibitors.

[0106] 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 CLDN6 but not GPC3 and a second substance that specifically antagonizes GPC3 but not antagonizes CLDN6. The first substance and the second substance can be separated, or they can be connected, fused or compounded. The CLDN6 antagonist can be a first CAR having an extracellular antigen binding domain that binds CLDN6 but not GPC3, and the GPC3 antagonist can be a second CAR having an extracellular antigen binding domain that binds GPC3 but not CLDN6, and the first CAR and the second CAR can be separate polypeptides or connected together in the same chain. The CLDN6 antagonist can be a first group of engineered immune cells expressing a CAR that binds CLDN6 but not GPC3, and the GPC3 antagonist can be a second group of engineered immune cells expressing a CAR that binds GPC3 but not CLDN6. If the first substance and the second substance can be connected or fused together as a new molecule that retains the antagonistic ability of the two substances, the new molecule can also be considered as an "antagonist of CLDN6 and GPC3".

[0107] As used herein, the term "antagonist of CLDN6 and GPC3" refers to a substance that has the ability to specifically antagonize CLDN6 and GPC3 simultaneously. The antagonist of CLDN6 and GPC3 may be a CAR having an extracellular antigen binding domain capable of binding to CLDN6 and GPC3. The antagonist of CLDN6 and GPC3 may be an engineered immune cell expressing a CAR having an extracellular antigen binding domain capable of binding to CLDN6 and GPC3. The antagonist of CLDN6 and GPC3 may be an engineered immune cell expressing a first CAR and a second CAR, the first CAR having an extracellular antigen binding domain that binds to CLDN6 but not to GPC3, and the second CAR having 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.

[0108] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or related symptoms (eg, diabetes or cancer).

[0109] As used herein, "delaying" the development of cancer refers to postponing, hindering, slowing down, slowing down, stabilizing and / or delaying the development of the disease. The delay can have different time lengths, depending on the history of the disease and / or the individual being treated. It is obvious to those skilled in the art that sufficient or significant delays can actually include prevention because the individual does not suffer from the disease. Compared with not using the method, the method of "delaying" the development of cancer is a method of reducing the possibility of disease development within a given time frame and / or reducing the degree of disease within a given time frame. Such comparisons are generally based on clinical studies conducted using a statistically significant number of individuals. Standard methods can be used to detect cancer development, which include but are not limited to computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography or biopsy. Development can also refer to cancer progression that may not be detected initially, and includes occurrence, recurrence and onset.

[0110] As used herein, "Claudin 6-related disease or disorder" refers to a disease or disorder involving 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 on which Claudin 6 is abnormally expressed. A Claudin 6-related disease or disorder may include cells in which or on which at least one activity of Claudin 6 is defective. In some embodiments, a Claudin 6-related disease or disorder is cancer, such as a solid tumor cancer.

[0111] As used herein, "GPC3-associated disease or disorder" refers to a disease or disorder involving cells or tissues in which GPC3 is expressed or overexpressed. A GPC3-associated disease or disorder may include cells on which GPC3 is abnormally expressed. A GPC3-associated disease or disorder may include cells in which or on which at least one activity of GPC3 is defective. In some embodiments, the GPC3-associated disease or disorder is cancer, such as a solid tumor cancer.

[0112] The terms "about" and "approximately" mean 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.

[0113] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0114] It should be understood that anywhere in this document an embodiment is described using the term "comprising", similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that anywhere in this document an embodiment is described using the phrase "consisting essentially of", similar embodiments described with "consisting of" are also provided.

[0115] The term “between” used in the phrase “between A and B” or “between AB” means a range including A and B.

[0116] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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).

[0117] 5.2. Single domain antibodies

[0118] 5.2.1. Single domain antibodies binding to claudin 6

[0119] In one aspect, provided herein are single domain antibodies (eg, VHH domains) capable of binding to Claudin 6.

[0120] In some embodiments, a single domain antibody (eg, a VHH domain) provided herein binds to human Claudin 6. CLDN6 (UniProtKB: P56747) is a four-transmembrane protein involved in the formation of tight junctions during development.

[0121] The anti-Claudin 6 single domain antibodies provided herein can modulate one or more activities of Claudin 6. In some embodiments, the anti-Claudin 6 single domain antibodies provided herein are antagonist antibodies.

[0122] The anti-Claudin 6 single domain antibodies provided herein can be used at ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (K D) binds to claudin 6 (e.g., human claudin 6). A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure, including by RIA, for example, with a Fab form of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays; by Use e.g. Red96 system; or through Use e.g. TM-2000 or TM-3000. The "association rate" or "kon" can also be measured using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, using e.g. Red96, TM-2000 or TM-3000 system for determination.

[0123] In some embodiments, the anti-claudin 6 single domain antibodies provided herein are VHH domains. The exemplary VHH domains provided herein are generated as described in Section 6 below, and these VHH domains are referred to as 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1, and 77LICNB02H2.

[0124] Therefore, in some embodiments, the single domain antibody provided herein comprises one or more CDR sequences of any one of 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1 and 77LICNB02H2. In some embodiments, a single domain antibody that binds to Claudin 6 comprising the following structure is provided herein: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequence is selected from those of 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1 and 77LICNB02H2.

[0125] In some embodiments, an anti-claudin 6 single domain antibody comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO: 7 is provided. In some embodiments, an anti-claudin 6 single domain antibody comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO: 8 is provided. In some embodiments, an anti-claudin 6 single domain antibody comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO: 9 is provided. In some embodiments, an anti-claudin 6 single domain antibody comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO: 10 is provided. In some embodiments, an anti-claudin 6 single domain antibody comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO: 11 is provided. In some embodiments, the anti-claudin 6 single domain antibody is of camelids. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0126] In some embodiments, a single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 7. In some embodiments, a single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 7. In other embodiments, a single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 7. In some embodiments, a single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 7. In some embodiments, a single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 7. In some embodiments, a single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 7. In some embodiments, the single domain antibody has CDR1, CDR2 and CDR3, which have the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO:7. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDR is determined according to the IMGT numbering scheme. In some embodiments, the CDR is determined according to the Kabat numbering scheme. In some embodiments, the CDR is determined according to the AbM numbering scheme. In other embodiments, the CDR is determined according to the Chothia numbering scheme. In other embodiments, the CDR is determined according to the Contact numbering scheme. The CDR can be determined according to a combination of any of the above numbering schemes. In some embodiments, the anti-claudin 6 single domain antibody is of camelids. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody can comprise an acceptor human framework, such as a human immunoglobulin framework or a human common framework.

[0127] In some embodiments, a single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 8. In some embodiments, a single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 8. In other embodiments, a single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 8. In some embodiments, a single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 8. In some embodiments, a single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 8. In some embodiments, a single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 8. In some embodiments, the single domain antibody has CDR1, CDR2 and CDR3, which have the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO:8. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDR is determined according to the IMGT numbering scheme. In some embodiments, the CDR is determined according to the Kabat numbering scheme. In some embodiments, the CDR is determined according to the AbM numbering scheme. In other embodiments, the CDR is determined according to the Chothia numbering scheme. In other embodiments, the CDR is determined according to the Contact numbering scheme. The CDR can be determined according to a combination of any of the above numbering schemes. In some embodiments, the anti-claudin 6 single domain antibody is of camelids. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human common framework.

[0128] In some embodiments, a single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 9. In some embodiments, a single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 9. In other embodiments, a single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 9. In some embodiments, a single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 9. In some embodiments, a single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 9. In some embodiments, a single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 9. In some embodiments, the single domain antibody has CDR1, CDR2 and CDR3, which have the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO:9. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDR is determined according to the IMGT numbering scheme. In some embodiments, the CDR is determined according to the Kabat numbering scheme. In some embodiments, the CDR is determined according to the AbM numbering scheme. In other embodiments, the CDR is determined according to the Chothia numbering scheme. In other embodiments, the CDR is determined according to the Contact numbering scheme. The CDR can be determined according to a combination of any of the above numbering schemes. In some embodiments, the anti-claudin 6 single domain antibody is of camelids. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human common framework.

[0129] In some embodiments, a single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 10. In some embodiments, a single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 10. In other embodiments, a single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 10. In some embodiments, a single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 10. In some embodiments, a single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 10. In some embodiments, a single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 10. In some embodiments, the single domain antibody has CDR1, CDR2 and CDR3, which have the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 10. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDR is determined according to the IMGT numbering scheme. In some embodiments, the CDR is determined according to the Kabat numbering scheme. In some embodiments, the CDR is determined according to the AbM numbering scheme. In other embodiments, the CDR is determined according to the Chothia numbering scheme. The CDR can be determined according to a combination of any of the above numbering schemes. In other embodiments, the CDR is numbered according to Contact. In some embodiments, the anti-claudin 6 single domain antibody is of camelids. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human common framework.

[0130] In some embodiments, a single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 11. In some embodiments, a single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 11. In other embodiments, a single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 11. In some embodiments, a single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 11. In some embodiments, a single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 11. In some embodiments, a single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 11. In some embodiments, the single domain antibody has CDR1, CDR2 and CDR3, which have the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 11. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDR is determined according to the IMGT numbering scheme. In some embodiments, the CDR is determined according to the Kabat numbering scheme. In some embodiments, the CDR is determined according to the AbM numbering scheme. In other embodiments, the CDR is determined according to the Chothia numbering scheme. In other embodiments, the CDR is determined according to the Contact numbering scheme. The CDR can be determined according to a combination of any of the above numbering schemes. In some embodiments, the anti-claudin 6 single domain antibody is of camelids. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human common framework.

[0131] In some embodiments, the present invention provides a single domain antibody that binds to tight junction protein 6 comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein (i) CDR1 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (ii) CDR2 comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; and / or (iii) CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the anti-tight junction protein 6 single domain antibody is of camelids. In some embodiments, the anti-tight junction protein 6 single domain antibody is humanized. The anti-tight junction protein 6 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0132] In other embodiments, the present invention provides a single-domain antibody that binds to Claudin 6, comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein (i) CDR1 comprises 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 to SEQ ID NO: 1 or SEQ ID NO: 2; (ii) CDR2 comprises 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 to SEQ ID NO: 3 or SEQ ID NO: 4; and / or (iii) CDR3 comprises 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 to SEQ ID NO: 5 or SEQ ID NO: 6; NO:5 or SEQ ID NO:6 has an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the anti-claudin 6 single domain antibody is of camelid. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0133] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 1; CDR2 comprises the amino acid sequence of SEQ ID NO: 3; and CDR3 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-claudin 6 single domain antibody is of camelid. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0134] 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 of camelid. In some embodiments, the anti-claudin 6 single domain antibody is humanized. The anti-claudin 6 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0135] 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 a sequence of SEQ ID NO: 7. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 8. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 9. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 10. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 11.

[0136] In some embodiments, the single domain antibodies provided herein are humanized single domain antibodies.Humanized single domain antibodies can be generated using the methods exemplified in Section 6 below or the methods described in the following sections.

[0137] The framework regions described herein are defined according to the boundaries of the CDR numbering system. In other words, if the CDRs are defined by, for example, Kabat, IMGT or Chothia, the framework regions are the amino acid residues surrounding the CDRs in the variable region in the form from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residue N-terminal to the CDR1 amino acid residue as defined by, e.g., 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 as defined by, e.g., 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 as defined by, e.g., 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 C-terminal to the CDR3 amino acid residue as defined by, e.g., the Kabat numbering system, the AbM numbering system, the IMGT numbering system, or the Chothia numbering system.

[0138] In some embodiments, an isolated anti-claudin 6 single domain antibody comprising a VHH domain having an amino acid sequence of SEQ ID NO: 7 is provided. In some embodiments, a polypeptide comprising an amino acid sequence of SEQ ID NO: 7 is provided. In some embodiments, an isolated anti-claudin 6 single domain antibody comprising a VHH domain having an amino acid sequence of SEQ ID NO: 8 is provided. In some embodiments, a polypeptide comprising an amino acid sequence of SEQ ID NO: 8 is provided. In some embodiments, an isolated anti-claudin 6 single domain antibody comprising a VHH domain having an amino acid sequence of SEQ ID NO: 9 is provided. In some embodiments, a polypeptide comprising an amino acid sequence of SEQ ID NO: 9 is provided. In some embodiments, an isolated anti-claudin 6 single domain antibody comprising a VHH domain having an amino acid sequence of SEQ ID NO: 10 is provided. In some embodiments, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10 is provided. In some embodiments, an isolated anti-claudin 6 single domain antibody comprising a VHH domain having an amino acid sequence of SEQ ID NO: 11 is provided. In some embodiments, a polypeptide comprising an amino acid sequence of SEQ ID NO: 11 is provided.

[0139] In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises an amino acid sequence having a certain percentage identity relative to any one of antibodies 77LICNB01, 77LICNB02, 77LICNB03, 77LICNB02H1, and 77LICNB02H2.

[0140] A mathematical algorithm can be used to determine the percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), modified as Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameter set, for example, for score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameter set, for example, for score 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. In order to obtain gap alignments for comparison purposes, Gapped BLAST as described in Altschul et al., Nucleic Acids Res. 25: 3389 3402 (1997) can be used. Alternatively, PSI BLAST can be used to perform an iterative search (Id.) for detecting distance relationships between molecules. When using BLAST, Gapped BLAST and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS 4: 11-17 (1998). This algorithm is incorporated in the ALIGN program (version 2.0), which is a part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table can be used, the gap length penalty is 12, and the gap penalty is 4. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating percent identity, typically only exact matches are counted.

[0141] In some embodiments, an anti-claudin 6 single domain antibody comprising a VHH domain is provided, 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 NO: 7-11. In some embodiments, the VHH sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains a substitution (e.g., conservative substitution), insertion or deletion relative 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, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence selected from SEQ ID NO: 7-11. In some embodiments, the substitution, insertion or deletion occurs in a region outside the CDR (i.e., in the FR). Optionally, the anti-claudin 6 single domain antibody comprises an amino acid sequence selected from SEQ ID NO: 7-11, including post-translational modifications of the sequence.

[0142] In certain 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 certain 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 certain 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 certain 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 certain embodiments, the single domain antibody described herein comprises 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.

[0143] Functional epitopes can be mapped, for example, by combined alanine scanning to identify amino acids in the claudin 6 protein that are necessary for interaction with the anti-claudin 6 single domain antibodies provided herein. The conformation and crystal structure of the anti-claudin 6 single domain antibodies that bind to claudin 6 can be used to identify epitopes. The present disclosure can provide an antibody that specifically binds to the same epitope as any anti-claudin 6 single domain antibody provided herein. For example, an antibody that binds to the same epitope as an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 7 is provided. An antibody that can bind to the same epitope as an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 8 is provided. An antibody that can bind to the same epitope as an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 9 is provided. An antibody that can bind to the same epitope as an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 10 is provided. An antibody that can bind to the same epitope as an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 11 is provided.

[0144] In some embodiments, the present invention provides an anti-claudin 6 antibody or an antigen-binding fragment thereof that specifically binds to claudin 6 competitively with any of the anti-claudin 6 single domain antibodies described herein. Competitive binding can be determined using an ELISA assay. For example, an antibody that specifically binds to claudin 6 competitively with an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 7 can be provided. An antibody that specifically binds to claudin 6 competitively with an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 8 can be provided. An antibody that specifically binds to claudin 6 competitively with an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 9 can be provided. An antibody that specifically binds to claudin 6 competitively with an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 10 can be provided. An antibody that specifically binds to claudin 6 competitively with an anti-claudin 6 single domain antibody comprising the amino acid sequence of SEQ ID NO: 11 can be provided.

[0145] In some embodiments, the present invention provides a tight junction protein 6 binding protein comprising any of the above-mentioned anti-tight junction protein 6 single domain antibodies. In some embodiments, the tight junction protein 6 binding protein is a monoclonal antibody, including camelid, chimeric, humanized or human antibodies. In some embodiments, the anti-tight junction protein 6 antibody is an antibody fragment, for example, a VHH fragment. In some embodiments, the anti-tight junction protein 6 antibody is a full-length heavy chain antibody containing only the Fc region of any antibody class or isotype (such as IgG1 or IgG4). The Fc region may have a reduced or minimized effector function. In some embodiments, the tight junction protein 6 binding protein is a fusion protein comprising an anti-tight junction protein 6 single domain antibody provided herein. The tight junction protein 6 binding protein can be a multispecific antibody comprising an anti-tight junction protein 6 single domain antibody provided herein. Other exemplary tight junction protein 6 binding molecules are described in more detail in the following sections.

[0146] In some embodiments, the anti-Claudin 6 antibody (such as an anti-Claudin 6 single domain antibody) or antigen binding protein according to any of the above embodiments may include any of the features, alone or in combination, as described in Sections 5.2.2 to 5.2.7 below.

[0147] 5.2.2. Humanized single domain antibodies

[0148] Single domain antibodies as described herein include humanized single domain antibodies. The general strategy for humanizing single domain antibodies from camelid species has been described (see, e.g., Vincke et al., J. Biol. Chem., 284 (5): 3273-3284 (2009)) and can be used to produce humanized VHH domains disclosed herein. The design of humanized single domain antibodies from camelid species can include hallmark residues in VHH, such as residues 11, 37, 44, 45 and 47 (residues numbered according to Kabat) (Muyldermans, Reviews Mol Biotech 74: 277-302 (2001)).

[0149] Humanized antibodies, such as the humanized single domain antibodies disclosed herein, can also be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. 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. Pat. No. 5,565,332) and, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res.55(23Supp):5973s-5977s(1995), Couto et al., Cancer Res.55(8):1717-22(1995), Sandhu JS, Gene 150(2):409-10(1994) and Pedersen et al. J. Mol. Biol.235(3):959-73(1994). See also U.S. Patent Publication No. US2005 / 0042664A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0150] In some embodiments, the single-domain antibodies provided herein may be humanized single-domain antibodies that bind to tight junction protein 6 (including human tight junction protein 6). For example, the humanized single-chain antibodies disclosed herein may include one or more CDRs shown in SEQ ID NO: 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 therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are typically taken from the "import" variable domain. Humanization can be performed, for example, by replacing the corresponding sequence of a human antibody with a hypervariable region sequence according to 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). Humanization of the single-domain antibodies provided herein is performed as described in Section 6 below.

[0151] In some cases, humanized antibodies are constructed by CDR transplantation, wherein the amino acid sequence of the CDR of the parent non-human antibody is transplanted onto the human antibody framework. For example, Padlan et al. determined that only about one-third of the residues in the CDR actually contact the antigen, and these are referred to as "specificity determining residues" or SDRs (Padlan et al., FASEB J.9:133-39 (1995)). In SDR transplantation technology, only SDR residues are transplanted onto the human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34 (2005)).

[0152] The human variable domains selected for the preparation of humanized antibodies may be important for reducing antigenicity. For example, according to the so-called "best fit" method, the sequence of the variable domains of non-human antibodies is screened for the entire library of known human variable domain sequences. The human sequence closest to non-human antibodies can be selected as the human framework of humanized antibodies (Sims et al., J.Immunol.151:2296-308 (1993) and Chothia et al., J.Mol.Biol.196:901-17 (1987)). Another method uses a specific framework of the consensus sequence of all human antibodies of a specific subgroup derived from a light chain or a heavy chain. 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 derived from the most abundant human subclass V L 6Subgroup I(V L 6I) and VH Subgroup III (V H In another approach, human germline genes are used as the source of framework regions.

[0153] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is irrelevant. The method includes comparing the non-human sequence with a functional human germline gene library. Then select genes that encode the same or closely related canonical structures as the mouse sequence. Next, among the genes that share a canonical structure with the non-human antibody, select those genes with the highest homology in the CDR as FR donors. Finally, non-human CDRs are transplanted onto these FRs (see, for example, Tan et al., J. Immunol. 169: 1119-25 (2002)).

[0154] It is also generally desirable to humanize antibodies to retain their affinity for antigens and other favorable biological properties. In order to achieve this goal, according to a method, humanized antibodies are prepared by using a three-dimensional model of the parent and humanized sequences to analyze the parent sequence and various conceptual humanized products. The three-dimensional immunoglobulin model is generally available and is familiar to those skilled in the art. A computer program that illustrates and displays the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequence can be obtained. 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)). Checking these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, such as analyzing the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for one or more target antigens, is achieved.In general, the hypervariable region residues are directly and most significantly involved in influencing antigen binding.

[0155] Another method of antibody humanization is based on an antibody human metric called Human String Content (HSC). The method compares mouse sequences with human germline gene repertoires and scores differences as HSC. The target sequence is then humanized to generate a variety of different humanized variants by maximizing its HSC rather than using a global identity measurement (Lazar et al., Mol. Immunol. 44: 1986-98 (2007)).

[0156] 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 humanized variant libraries and the use of enrichment techniques or high-throughput screening techniques to select the best clones. Antibody variants can be isolated from phage, ribosome and yeast display libraries and 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)).

[0157] In the FR library method, a batch of residue variants are introduced at specific positions in the FR, and the library is then screened to select the FR that best supports the transplanted CDR. The residues to be replaced can include some or all of the "vernier" residues identified as potentially contributing to the CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-99 (1992)), or a more limited set of target residues identified by Baca et al. J. Biol. Chem. 272: 10678-84 (1997).

[0158] In FR shuffling, the entire FR is combined with a non-human CDR rather than creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., Methods 36:43-60 (2005)). A one-step FR shuffling process can be used. This approach has been shown to be effective because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60 (2007)).

[0159] The "humanization engineering" (humaneering) method is based on the experimental identification of the essential minimum specific determinant (MSD) and on the sequential substitution of non-human fragments into a human FR library and the assessment of binding. This method generally results in epitope preservation and the identification of antibodies from multiple subclasses with different human V segment CDRs.

[0160] "Human engineering" (human engineering) methods involve changing non-human antibodies or antibody fragments by making specific changes to the amino acid sequence of the antibody, thereby producing a modified antibody with reduced immunogenicity in humans, which still retains the desired binding properties of the original non-human antibody. Generally, the technology involves classifying the amino acid residues of non-human antibodies as "low risk", "medium risk" or "high risk" residues. Classification is performed using an overall risk / reward calculation, which calculates the predicted benefit of performing a specific substitution (e.g., for human immunogenicity) and the risk that the substitution will affect the folding of the resulting antibody. The specific human amino acid residues that will be substituted at a given position (e.g., low risk or medium risk) of the non-human antibody sequence can be selected by comparing the amino acid sequence from the variable region of the non-human antibody with the corresponding region of a specific or shared human antibody sequence. The amino acid residues at low risk or medium risk positions in the non-human sequence can replace the corresponding residues in the human antibody sequence according to the comparison. Techniques for making human engineered proteins are described in more detail in Studnicka et al., Protein Engineering 7:805-14 (1994); US Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619; and PCT Publication WO 93 / 11794.

[0161] Composite Human Antibody can be used, for example TM Composite human antibodies were produced using the Antitope technology (Antitope Ltd., Cambridge, United Kingdom). To produce composite human antibodies, variable region sequences were designed from fragments of multiple human antibody variable region sequences in a way that avoided T cell epitopes, thereby minimizing the immunogenicity of the resulting antibodies.

[0162] Deimmunized antibodies are antibodies in which T cell epitopes have been removed. Methods for preparing deimmunized antibodies have been 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). Deimmunized antibodies comprise variable regions and human constant regions in which T cell epitopes are missing. In brief, the variable regions of the antibodies are cloned, and then T cell epitopes are identified by testing overlapping peptides derived from the variable regions of the antibodies in T cell proliferation assays. T cell epitopes are identified by computer methods to identify peptides that bind to human MHC class II. Mutations are introduced into the variable regions to eliminate binding to human MHC class II. The mutated variable regions are then used to generate deimmunized antibodies.

[0163] 5.2.3. Single domain antibody variants

[0164] In some embodiments, one or more amino acid sequence modifications of the single-domain antibodies that bind to tight junction protein 6 described herein are contemplated. For example, it may be necessary to optimize the binding affinity and / or other biological properties of the antibody, 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 tight junction protein 6 described herein, it is contemplated that variants of the single-domain antibodies that bind to tight junction protein 6 described herein can also be prepared. For example, single-domain antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art understand that amino acid changes can change the post-translational process of single-domain antibodies.

[0165] Chemical modification

[0166] In some embodiments, the single domain antibodies provided herein are chemically modified, for example, by covalently attaching any type of molecule to a single domain antibody. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to cell ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., a portion of Fc or Fc). Any of a variety of chemical modifications may be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In addition, the antibody may contain one or more non-classical amino acids.

[0167] The antibodies provided herein can be altered to increase or decrease the degree of antibody glycosylation. The addition or deletion of antibody glycosylation sites can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites.

[0168] When the single domain antibody provided herein is fused to the Fc region, the carbohydrate connected thereto can be changed. The natural antibody produced by mammalian cells generally comprises a branched biantennary oligosaccharide, which is generally connected to the Asn297 of the CH2 domain of the Fc region by an N bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose connected to GlcNAc in the "stem" of the biantennary oligosaccharide structure. The oligosaccharides in the binding molecules provided herein can be modified to produce variants with certain improved properties.

[0169] In other embodiments, when the single domain antibody provided herein is fused to the Fc region, the antibody variant provided herein may have a carbohydrate structure lacking fucose (directly or indirectly) connected to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn 297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high mannose structures) connected to Asn297 as measured by MALDI-TOF mass spectrometry, as described in, for example, WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence changes in antibodies, Asn297 may also be located at approximately ±3 amino acids upstream or downstream of position 297, i.e., between position 294 and position 300. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US2003 / 0157108 and US2004 / 0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO2005 / 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 defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108; and WO2004 / 056312, particularly at Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0170] The binding molecules comprising the single domain antibodies provided herein are further provided with bisected oligosaccharides, for example, wherein the biantennary oligosaccharides connected to the Fc region are bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.) and US2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharides connected to the Fc region are also provided. Such variants may have improved CDC function. Such variants are described in, for example, WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0171] In a molecule comprising a single domain antibody of the invention and an Fc region, one or more amino acid modifications may be introduced into the Fc region to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0172] The present application contemplates variants with some (but not all) effector functions that make the antibody variant an ideal candidate for applications where the in vivo half-life of the binding molecule is important but certain effector functions (such as complement and ADCC) are non-essential or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the binding molecule has no FcγR binding ability (and therefore may lack ADCC activity), but may retain FcRn binding ability. In U.S. Pat. Nos. 5,500,362 (see, e.g., 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)), non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described. Alternatively, non-radioactive assays may be employed, see, e.g., ACTI for flow cytometry. TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., 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 determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0173] Binding molecules with reduced effector function include antibodies that replace one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants that replace two or more of amino acid positions 265, 269, 270, 297, and 327, including an Fc mutant called "DANA" in which residues 265 and 297 are replaced by alanine (U.S. Pat. No. 7,332,581).

[0174] Certain variants with increased or decreased binding to FcRs are 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)).

[0175] The variant may comprise an Fc region having one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 within the Fc region (EU numbering of residues). Alterations within the Fc region may occur that result in altered (i.e., increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0176] Binding molecules with 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 US 2005 / 0014934A1 (Hinton et al.). Those molecules comprise an Fc region with one or more amino acid substitutions, wherein these substitutions improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0177] It may be desirable to generate cysteine ​​engineered antibodies in which one or more residues of an antibody are substituted with cysteine ​​residues. The substituted residues may occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein.

[0178] Substitution, deletion or insertion

[0179] The mutation can be a substitution, deletion or insertion of one or more codons encoding a single domain antibody or polypeptide, resulting in a change in the amino acid sequence compared to the original antibody or polypeptide. The target sites for substitution mutagenesis include CDR and FR.

[0180] Amino acid substitution can 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 conservative amino acid substitution. Standard techniques known to those skilled in the art can be used for introducing mutations in the nucleotide sequence encoding the molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that cause amino acid substitutions. Insertion or deletion can be optionally within the range of about 1 to 5 amino acids. Substitution, deletion or insertion can 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. Substitution is a conservative amino acid substitution that can be performed at one or more predicted non-essential amino acid residues. The allowed changes can be determined by systematically performing amino acid insertions, deletions or substitutions in the sequence and testing the activity exhibited by the parent antibody of the resulting variant.

[0181] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue.

[0182] The present disclosure includes single domain antibodies generated by conservative amino acid substitution. In conservative amino acid substitution, amino acid residues are replaced by amino acid residues with side chains with similar charges. As described above, families of amino acid residues with side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coded sequence, for example, by saturation mutagenesis, and the biological activity of the resulting mutant can be screened to identify the mutant retaining activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. Conservative (for example, in an amino acid group with similar properties and / or side chains) substitutions can be carried out to maintain or not significantly change properties. Exemplary substitutions are shown in Table 2 below.

[0183] Table 2. Amino acid substitutions

[0184] Amino acids can be grouped according to the similarity of the properties of their side chains (see, e.g., 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 according to 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 that is not involved in maintaining the correct conformation of the single domain antibody can also be substituted, for example, with another amino acid such as alanine or serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Non-conservative substitutions will require exchanging a member of one of these categories for another category.

[0185] A substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, one or more resulting variants selected for further study will have modifications (e.g., improvements) relative to certain biological properties (e.g., increased affinity, reduced immunogenicity) of the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity mature antibodies that can be conveniently generated, for example, using affinity maturation techniques based on phage display (such as those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0186] Changes (e.g., substitutions) can be made in CDRs, for example, to improve antibody affinity. Such changes can be made in CDR "hot spots", i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and / or SDRs (a-CDRs), wherein the binding affinity of the resulting variant antibody or fragment thereof is tested. Affinity maturation by construction and reselection from a secondary library is described in, e.g., Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., 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-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves a CDR-guided approach in which several CDR residues (e.g., 4-6 residues at a time) are randomized. The CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. The following section provides a more detailed description of affinity maturation.

[0187] Substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. In some embodiments of the variant VHH sequences provided herein, each CDR is unchanged or contains no more than one, two or three amino acid substitutions.

[0188] As described in Cunningham and Wells, Science, 244: 1081-1085 (1989), a useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is called "alanine scanning mutagenesis". In this method, the residue or residue group of the target residue (e.g., charged residues such as Arg, Asp, His, Lys and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be introduced at the amino acid position to demonstrate functional sensitivity to the initial substitution. 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 or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.

[0189] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions, ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusing the N-terminus or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antigen-binding domain antibody.

[0190] Methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning and PCR mutagenesis can be used to make changes. Site-directed 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 can be performed on cloned DNA to generate single domain antibody variant DNA.

[0191] 5.2.4. In vitro affinity maturation

[0192] Antibody variants with improved properties such as affinity, stability or expression level compared to the parent antibody can be prepared by in vitro affinity maturation.As with the natural prototype, in vitro affinity maturation is based on the principle of mutation and selection.Antibody libraries are displayed on the surface of an organism (e.g., phage, bacterium, yeast or mammalian cell) or associated (e.g., covalently or non-covalently) with its encoding mRNA or DNA.The affinity selection of displayed antibodies allows the separation of an organism or a complex carrying the genetic information of the encoding antibody.Use display methods such as two or three rounds of mutation and selection of phage display to produce antibody fragments with affinity in the low nanomolar range.Antibodies of affinity maturation can have nanomolar or even picomolar affinity to the target antigen.

[0193] 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 involves exposure to antigens to allow phage-displayed antibodies to bind to their targets, a process known as "panning". Phages bound to antigens are recovered and used to infect bacteria to produce phages for further rounds of selection. For review, see, for example, Hoogenboom, Methods. Mol. Biol. 178: 1-37 (2002) and Bradbury and Marks, J. Immunol. Methods 290: 29-49 (2004).

[0194] In a yeast display system (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997) and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can be fused to the adhesion subunit of the yeast lectin protein Aga2p, which is attached to the yeast cell wall via a disulfide bond to Aga1p. Displaying the protein by Aga2p protrudes the protein from the cell surface, thereby minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen the library to select antibodies with improved affinity or stability. Binding to a soluble antigen of interest is determined by labeling the yeast with a biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Changes in antibody surface expression can be measured by immunofluorescence labeling of hemagglutinin or c-Myc epitope tags (e.g., scFv) flanking single-chain antibodies. Expression has been shown to correlate with the stability of the displayed protein, and therefore antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., J. Mol. Biol. 292:949-56 (1999)). Another advantage of yeast display is that the displayed protein is folded in the endoplasmic reticulum of eukaryotic yeast cells, utilizing endoplasmic reticulum chaperones and quality control mechanisms. Once maturation is complete, antibody affinity can be conveniently "titrated" while displayed on the yeast surface, thereby eliminating the need to express and purify each clone. A theoretical limitation of yeast surface display is the smaller functional library size that is possible with other display methods; however, recent methods use the mating system of yeast cells to produce libraries estimated to be 10 14 of combinatorial diversity (see, e.g., U.S. Patent Publication 2003 / 0186374 and Blaise et al., Gene 342:211-18 (2004)).

[0195] In ribosome display, antibody-ribosome-mRNA (ARM) complex is produced for selection in a cell-free system. The DNA library encoding the specific antibody library is fused with the spacer sequence gene lacking a stop codon. The spacer sequence is still connected to the peptidyl tRNA during translation and occupies the ribosome channel, so that the target protein is protruded and folded from the ribosome. The resulting mRNA, ribosome and protein complex can be combined with a surface-bound ligand, thereby allowing the antibody and its encoding mRNA to be separated simultaneously by affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back to cDNA, which can then be mutagenized and used for 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 antibody and mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98: 3750-55 (2001)).

[0196] Since these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the efficiency of transformation of the bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be easily introduced after each round of selection, for example, by non-proofreading polymerases, since no library has to be transformed after any diversification step.

[0197] A mammalian display system may be used.

[0198] Diversity can also be introduced into the CDR of the antibody library in a targeted manner or by random introduction. The former method includes all CDRs of the antibody or the hot spots of the separation of the targeted somatic hypermutation by high-level or low-level mutagenesis sequence (see, for example, Ho et al., J.Biol.Chem.280:607-17 (2005)) or residues suspected of affecting affinity based on experimental or structural reasons. Diversity can also be introduced by replacing the naturally diverse regions by DNA shuffling or similar techniques (see, for example, Lu et al., J.Biol.Chem.278:43496-507 (2003); U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative technology targets hypervariable loops extending into framework region residues (see, for example, Bond et al., J.Mol.Biol.348:699-709 (2005)), using loop deletions and insertions in CDR or using hybridization-based diversification (see, for example, U.S. Patent Publication No. 2004 / 0005709). Other methods for generating diversity in CDRs are disclosed in, for example, U.S. Pat. No. 7,985,840. Additional methods for generating antibody libraries and / or antibody affinity maturation are disclosed in, for example, 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, each of which is incorporated herein by reference.

[0199] Screening of the library can be accomplished by various techniques known in the art. For example, the single domain antibody can be immobilized on a solid support, column, needle, or cellulose / poly(vinylidene fluoride) membrane / other filter, expressed on a host cell attached to an adsorption plate or used for cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method to pan the display library.

[0200] For review of in vitro affinity maturation methods, see, eg, Hoogenboom, Nature Biotechnology 23: 1105-16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4: 39-51 (2010); and references therein.

[0201] 5.2.5. Modification of single domain antibodies

[0202] Covalent modification of single domain antibodies is included within the scope of the present disclosure. Covalent modification includes reacting targeted amino acid residues of the single domain antibody with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues of the single domain antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, hydroxyphosphorylation of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (see, e.g., Creighton, et al., 2001). Proteins:Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0203] Other types of covalent modifications of single domain antibodies included within the scope of the present disclosure include altering the native glycosylation pattern of the antibody or polypeptide as described above (see, e.g., Beck et al., Curr. Pharm. Biotechnol. 9:482-501 (2008); and Walsh, Drug Discov. Today 15:773-80 (2010)), and linking the antibody to one of a variety of non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene, in a manner such as described in U.S. Pat. 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 the claudin 6 of the present disclosure can also be genetically fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) to extend half-life and / or confer known Fc-mediated effector functions.

[0204] The single-chain antibody binding to Claudin 6 of the present disclosure can also be modified to form chimeric molecules comprising a polypeptide or amino acid sequence conjugated 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 (Chamow and Ashkenazi, eds., 1999)) fused to a single-chain antibody that binds to Claudin 6. The single-chain antibody that binds to Claudin 6 can also be used to generate a chimeric antigen receptor (CAR) that binds to Claudin 6, as described in more detail below.

[0205] Also provided herein is a fusion protein comprising: a single-chain antibody that binds to the disclosed Claudin 6, and a heterologous polypeptide. In some embodiments, a heterologous polypeptide genetically fused or chemically conjugated to an antibody can be used to target the antibody to cells with Claudin 6 expressed on the cell surface.

[0206] Also provided herein are antibody sets that bind to the claudin 6 antigen. The antibody sets may have different on-rates, different off-rates, different affinities for the claudin 6 antigen, and / or different specificities for the claudin 6 antigen. The set may contain or consist of about 10 to about 1000 or more antibodies. The antibody set may be used, for example, in a 96-well or 384-well plate for assays such as ELISA.

[0207] 5.2.6. Preparation of single domain antibodies

[0208] Methods for preparing single domain antibodies have been described. See, for example, Els Pardon et al., Nature Protocol, 9(3):674 (2014). Single domain antibodies (such as VHH) can be obtained using methods known in the art, for example, by immunizing a camelid species (such as a camel or llama) and obtaining a hybridoma therefrom, or by cloning a single domain antibody library using molecular biology techniques known in the art and then selecting by ELISA with a single clone of an unselected library or by using phage display.

[0209] The single-domain antibodies provided herein can be produced by culturing cells transformed or transfected with a vector containing a nucleic acid encoding a single-domain antibody. The polynucleotide sequences encoding the polypeptide components of the antibodies disclosed herein can be obtained using standard recombinant techniques. The desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells such as hybridoma cells or B 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 heterologous polynucleotides in a host cell. Many vectors available and known in the art can be used for the purposes of the present disclosure. The selection of a suitable vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Host cells suitable for expressing the antibodies disclosed herein include prokaryotes such as archaea and eubacteria, including Gram-negative or Gram-positive organisms, eukaryotic microorganisms such as filamentous fungi or yeast, invertebrate cells such as insects or plant cells, and vertebrate cells such as mammalian host cell lines. The host cells are transformed with the above-mentioned expression vectors and cultured in conventional nutrient media, which are appropriately modified to induce promoters, select transformants, or amplify genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0210] Methods for antibody production including vector construction, expression and purification are further described in: Plückthun et al., Antibody Engineering: Producing antibodies in Escherichiacoli: From PCR to fermentation203-52 (McCafferty et al., eds., 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 FabFragments in Escherichia coli, Antibody Expression and Production (Al-Rubeai ed., 2011); and Therapeutic Monoclonal Antibodies:From Bench to Clinic (An ed., 2009).

[0211] Of course, it is contemplated that alternative methods well known in the art may be employed to prepare anti-Claudin 6 single domain antibodies. For example, the appropriate amino acid sequence or portion thereof may be produced by direct peptide synthesis using solid phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis (1969) and Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963). In vitro protein synthesis can be performed using manual techniques or by automation. The various parts of the anti-claudin 6 antibody can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-claudin 6 antibody. Alternatively, the antibody can be purified from cells or body fluids such as milk of transgenic animals engineered to express the antibody, as disclosed in U.S. Pat. Nos. 5,545,807 and 5,827,690.

[0212] Specifically, the single domain antibodies or other claudin 6 binding proteins provided herein can be produced by immunizing llamas, sorting single B cells, extracting V genes, cloning claudin 6 binding proteins (such as VHH-Fc fusions), and then expressing and purifying them on a small scale. Additional screening can also be performed on single domain antibodies and other molecules that bind to claudin 6, including selecting ELISA-positive, BLI-positive and K D One or more of less than 100 nM. These selection criteria can be combined as described in Section 6 below. In addition, the ability of individual VHH binding proteins (and other molecules that bind to tight junction protein 6) to bind to cells expressing tight junction protein 6 can be determined. Such determinations can be performed using FACS analysis of cells expressing tight junction protein 6 and measuring the mean fluorescence intensity (MFI) of fluorescently labeled VHH molecules. Each of the above aspects will be described in more detail below.

[0213] Polyclonal antibodies

[0214] Polyclonal antibodies are usually raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR (where R and R 1 The relevant antigen is conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin or soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic mycobacterium trehalose ester). The immunization protocol can be selected by those skilled in the art without undue experimentation.

[0215] For example, animals are immunized against antigens, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 of the original amount of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, the animals are bled and the antibody titer of the serum is determined. The animals are boosted until the titer plateaus. The conjugates can also be made into protein fusions in recombinant cell cultures. In addition, aggregating agents such as alum are suitable for enhancing immune responses.

[0216] Monoclonal antibodies

[0217] A monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0218] For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (US Pat. No. 4,816,567).

[0219] In the hybridoma method, a suitable host animal is immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0220] Immunizing agents typically include antigenic proteins or fusion variants thereof. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103. Immortalized cell lines are typically transformed mammalian cells. The hybridoma cells thus prepared are inoculated and grown in a suitable culture medium that may contain one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Preferred immortalized myeloma cells are those that fuse efficiently, support the stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a culture medium such as HAT culture medium.

[0221] The production of monoclonal antibodies against the antigen in the culture medium in which the hybridoma cells are grown is determined. The presence of monoclonal antibodies against the desired antigen in the culture medium in which the hybridoma cells are cultured can be determined. Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0222] After identifying hybridoma cells that produce antibodies with desired specificity, affinity and / or activity, clones can be subcloned and grown by standard methods (Goding above) by limiting dilution procedures. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 culture media. In addition, hybridoma cells can be grown in mammals as tumors.

[0223] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0224] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as described in U.S. Patent No. 4,816,567 and as described above. DNA encoding monoclonal antibodies is easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). Hybridoma cells are used as the preferred source of such DNA. Once separated, DNA can be placed in an expression vector, which is then transfected into host cells such as Escherichia coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins, to synthesize monoclonal antibodies in such recombinant host cells. Review articles on recombinantly expressing DNA encoding antibodies in bacteria include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pliickthun, Immunol. Revs. 130: 151-188 (1992).

[0225] 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 describe strategies for constructing very large phage libraries by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)) and combinatorial infection and in vivo recombination to produce high affinity (nM range) human antibodies (Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0226] The DNA may also be modified, for example, by replacing the coding sequence (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently linking to the coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides may be substituted to produce a chimeric bivalent antibody comprising one antigen binding site specific for an antigen and another antigen binding site specific for a different antigen.

[0227] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry (including those involving cross-linking agents). For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0228] Recombinant production in prokaryotes

[0229] The polynucleotide sequence encoding the antibody disclosed herein can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells such as hybridoma cells. Alternatively, a nucleotide synthesizer or PCR technique can be used to synthesize polynucleotides. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purpose of the present disclosure. The selection of suitable vectors will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains different components, depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it is located. Vector components generally 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.

[0230] Typically, plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in combination with these hosts. The vector typically carries a replication site, and a marker sequence that can provide phenotypic selection in the transformed cell. For example, Escherichia coli is typically transformed using pBR322 (a plasmid derived from an Escherichia coli species). Examples of pBR322 derivatives for expressing specific antibodies are described in detail in the U.S. Patent No. 5,648,237 of Carter et al.

[0231] In addition, phage vectors containing replicons and control sequences that are compatible with the host microorganism can be used as transformation vectors associated with these hosts. For example, phage such as GEM TM -11 can be used to prepare a recombinant vector, which can be used to transform susceptible host cells such as Escherichia coli LE392.

[0232] The expression vector of the present application can comprise two or more promoter-cistron pairs, encoding every kind of polypeptide component.Promoter is the non-translated regulatory sequence that is located at the upstream (5') of the cistron that regulates its expression.Prokaryotic promoters are generally divided into two categories, inducible and constitutive.Inducible promoter is the promoter that initiates the increased transcription level of the cistron under its control in response to the change of culture conditions (for example, the presence or absence of nutrients, or the change of temperature).

[0233] A large number of promoters recognized by a variety of potential host cells are well known. The promoter selected can be operably connected 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 separated promoter sequence into the vector of the present application. Natural promoter sequences and many heterologous promoters can be used to instruct the amplification and / or expression of the target gene. Heterologous promoters can be used because they usually allow greater transcription and higher yield of the target gene expressed compared with the natural target polypeptide promoter.

[0234] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, β-galactosidase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as tac or trc promoters. However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleic acid sequences have been disclosed so that the skilled person can operably link them to the cistron encoding the target peptide using a linker or adapter (Siebenlist et al. Cell 20: 269 (1980)) to provide any desired restriction sites.

[0235] On the one hand, each cistron in the recombinant vector comprises a secretory signal sequence component that instructs the translocation of the expressed polypeptide across the membrane. Generally, the signal sequence can be a component of the vector, or it can be a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purpose of the present disclosure should be a signal sequence that can be recognized and processed (i.e., cut by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the natural signal sequence of heterologous polypeptides, the signal sequence can be replaced by 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.

[0236] Production of antibodies according to the present disclosure can occur in the cytoplasm of the host cell and therefore does not require the presence of a secretion signal sequence within each cistron. Certain host strains (e.g., E. coli trxB - strains) provide cytoplasmic conditions that are favorable for disulfide bond formation, thereby allowing the expressed protein subunits to fold and assemble correctly.

[0237] Prokaryotic host cells suitable for expressing antibodies of the present disclosure include archaea and true bacteria, such as gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla or Paracoccus. In certain embodiments, gram-negative cells are used. Escherichia coli cells can be used as hosts. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325) and derivatives thereof, including strains having the genotype W3110AfhuA(AtonA)ptr3lacIqlacL8AompTA(nmpc-fepE)degP41kan R The strain 33D3 (U.S. Patent No. 5,639,635) of Escherichia coli is also suitable. Other strains and derivatives thereof, such as Escherichia coli 294 (ATCC 31,446), Escherichia coli B, Escherichia coli 1776 (ATCC 31,537) and Escherichia coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and not restrictive. Methods for constructing derivatives of any of the above-mentioned bacteria with a determined genotype are known in the art and are described in, for example, Bass et al., Proteins, 8:309-314 (1990). Considering the reproducibility of the replicon in bacterial cells, it is generally necessary to select suitable bacteria. For example, when a well-known plasmid (such as pBR322, pBR325, pACYC177 or pKN410) is used to supply the replicon, Escherichia coli, Serratia or Salmonella species can be suitably used as hosts.

[0238] Typically, the host cells should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may ideally be incorporated into the cell culture.

[0239] Host cells are transformed with the above-mentioned expression vectors and cultured in conventional nutrient media, which are suitably modified to induce promoters, select transformants or amplify genes encoding the desired sequence. Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can be replicated as an extrachromosomal element or by a chromosomal integrant. Depending on the host cell used, standard techniques suitable for these cells are used for transformation. Calcium treatment using calcium chloride is generally used for bacterial cells containing a large amount of cell wall barriers. Another method of transformation uses polyethylene glycol / DMSO. Another technique used is electroporation.

[0240] Prokaryotic cells used to produce antibodies of the present application are grown in culture media known in the art and suitable for culturing selected host cells. Examples of suitable culture media include Luria broth (LB) plus essential nutritional supplements. The culture media may also contain a selection agent selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture media to grow cells expressing an ampicillin resistance gene.

[0241] Any necessary supplements except carbon, nitrogen and inorganic phosphate sources may also be introduced separately or as a mixture with another supplement or medium such as a complex nitrogen source at appropriate concentrations. Optionally, the culture medium may contain one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol. Prokaryotic host cells are cultivated at a suitable temperature and pH.

[0242] If 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 transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-limited medium for induction. For example, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods 263: 133-147 (2002)). Depending on the vector construct used, a variety of other inducers known in the art can be used.

[0243] The expressed antibodies of the present disclosure are secreted into the periplasm of the host cell and recovered therefrom. Protein recovery generally involves destroying microorganisms, generally by methods such as osmotic shock, ultrasonic treatment or lysis. Once the cells are destroyed, cell debris or intact 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 to a culture medium and separated therein. Cells can be removed from the culture, and the culture supernatant can be filtered and concentrated to further purify the protein produced. The expressed polypeptide can be further separated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting assays.

[0244] Alternatively, protein production is carried out in large quantities by fermentation methods. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. In order to improve the yield and quality of the antibodies disclosed herein, various fermentation conditions can be changed. For example, chaperone proteins have been shown to contribute to the correct folding and solubilization of heterologous proteins produced in bacterial host cells. Chen et al. J BioChem 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).

[0245] In order to minimize proteolysis of expressed heterologous proteins (especially those sensitive to proteolysis), certain host strains lacking proteolytic enzymes can be used in the present disclosure, such as, for example, U.S. Pat. No. 5,264,365; U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996). E. coli strains lacking proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins can be used as host cells in the expression system encoding the antibodies of the present application.

[0246] The antibodies produced herein can be further purified to obtain substantially homogeneous preparations for further determination and use. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica or cation exchange resins such as DEAE, chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75. Protein A fixed on a solid phase can, for example, be used in some embodiments for immunoaffinity purification of binding molecules disclosed herein. The solid phase for fixing protein A can be a column comprising a glass or silica surface, or a controlled microporous glass column or a silicic acid column. The column may have been coated with a reagent such as glycerol to try to prevent nonspecific adhesion of pollutants. The solid phase is then washed to remove pollutants that are nonspecifically bound to the solid phase. Finally, the target antibody is recovered from the solid phase by elution.

[0247] Recombinant production in eukaryotic cells

[0248] For eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0249] The vector for eukaryotic hosts can also be an insert of other polypeptides encoding signal sequences or having specific cleavage sites at the N-terminal of mature proteins or polypeptides. The selected heterologous signal sequence can be a heterologous signal sequence recognized and processed (i.e., cut by a signal peptidase) by a host cell. In mammalian cell expression, mammalian signal sequences and viral secretion leader sequences, such as herpes simplex gD signals, can be obtained. The DNA in this precursor region can be connected to the DNA encoding the application's antibody in the reading frame.

[0250] Typically, mammalian expression vectors do not require an origin of replication component (the SV40 origin is usually the only one that can be used because it contains the early promoter).

[0251] Expression and cloning vectors may contain selection genes, also known as selectable markers. Selection genes may encode proteins that confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline); complement auxotrophic deficiencies; or provide key nutrients that are not available from complex media.

[0252] An example of a selection scheme uses a drug to prevent the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and therefore survive the selection scheme. Examples of this type of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0253] Another example of suitable selective marker for mammalian cells is to identify cells that can take in nucleic acids encoding the present application's antibodies. For example, first, all transformants are cultured in a culture medium containing methotrexate (Mtx) (a competitive antagonist of DHFR) to identify cells transformed with the DHFR selection gene. When using wild-type DHFR, exemplary suitable host cells are Chinese hamster ovary (CHO) cell lines of DHFR activity defects. Alternatively, polypeptides, wild-type DHFR albumen and another selective marker such as aminoglycoside 3'-phosphotransferase (APH) transformed or co-transformed host cells (particularly wild-type hosts containing endogenous DHFR) of coding DNA sequences can be selected by cell growth in a culture medium containing a selection agent such as an aminoglycoside antibiotic for selective markers.

[0254] Expression and cloning vectors usually contain a promoter, which is recognized by the host organism and operably connected to the nucleic acid encoding the desired polypeptide sequence. Eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream of the transcription start site. Another sequence found at 70 to 80 bases upstream of the start of transcription of many genes can be included. The 3' end of most eukaryotic organisms can be a signal for adding a polyadenylic acid tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0255] Transcription of the polypeptide from the vector in a mammalian host cell can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40), heterologous mammalian promoters (e.g., actin promoters or immunoglobulin promoters), heat shock promoters, so long as these promoters are compatible with the host cell systems.

[0256] The transcription of the DNA encoding the antibody disclosed herein by higher eukaryotes is usually increased by inserting an enhancer sequence into a vector. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein and insulin) are now known. Examples include the SV40 enhancer (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer and the adenovirus enhancer located at the late side of the replication origin. Regarding the enhancing elements for activating eukaryotic promoters, see also Yaniv, Nature 297: 17-18 (1982). The enhancer can be spliced ​​into the vector at the 5' or 3' position of the polypeptide coding sequence, but is preferably located at the 5' site of the promoter.

[0257] Expression vectors for eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain sequences required to terminate transcription and stabilize the mRNA. Such sequences are usually available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the polypeptide encoding the mRNA. A useful transcription termination component is the bovine growth hormone polyadenylation region.

[0258] Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryotic cells described herein, including vertebrate host cells.Propagation of vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney cell line (293 or 293 cell subclones for growth in suspension culture, 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 supporting 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 carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, 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 human hepatoma cell line (Hep G2).

[0259] Host cells can be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0260] Host cells for producing antibodies of the present application can be cultured in a variety of culture media. Commercially available culture 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, Ham et al., Meth.Enz.58:44 (1979); Barnes et al., Anal.Biochem.102:255 (1980); U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469; WO 90 / 03430; WO 87 / 00195; or any culture medium described in U.S. Pat. No. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed 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 TM Drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements at appropriate concentrations known to those skilled in the art may also be included. Culture conditions such as temperature, pH, and the like are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.

[0261] When using recombinant technology, the antibody can be produced in the intracellular, periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, the particle debris, host cells or cleavage fragments are removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the culture medium, usually first a commercially available protein concentration filter (such as Amicon or Millipore Pellicon ultrafiltration device) is used to concentrate the supernatant from this expression system. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants.

[0262] Protein compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The matrix to which the affinity ligand is connected is typically agarose, but other matrices are also available. Compared with agarose, mechanically stable matrices such as controlled pore glass or poly (styrene-divinyl) benzene allow faster flow rates and shorter processing times. Depending on the antibody to be recovered, other protein purification techniques can also be used, such as the heparin SEPHAROSE on the fractionation, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, anion or cation exchange resins (such as polyaspartic acid columns) on an ion exchange column. TM Chromatography, Chromatofocusing, SDS-PAGE and Ammonium Sulfate Precipitation Following any one or more of the initial purification steps, the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography.

[0263] 5.2.7. Binding molecules comprising single domain antibodies

[0264] In another aspect, provided herein is a binding molecule comprising a single domain antibody provided herein (e.g., a VHH domain to Claudin 6). In addition to the chimeric antigen receptor (CAR) provided herein as described in Section 5.3 below, in some embodiments, the single domain antibody to Claudin 6 provided herein is also part of other binding molecules. Exemplary binding molecules of the present disclosure are described herein.

[0265] Fusion Protein

[0266] In various embodiments, the single domain antibodies provided herein can be genetically 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 otherwise non-covalently conjugated to the agent. The agent can be a peptide or an antibody (or a fragment thereof).

[0267] Therefore, in some embodiments, provided herein are single domain antibodies (e.g., VHH domains) recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) to generate fusion proteins with heterologous proteins or polypeptides (or fragments thereof, e.g., polypeptides 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 more than 500 amino acids) and uses thereof. Specifically, provided herein are fusion proteins comprising antigen-binding fragments (e.g., CDR1, CDR2 and / or CDR3) of single domain antibodies provided herein and heterologous proteins, polypeptides or peptides.

[0268] In addition, the antibodies provided herein can be fused to a marker or "tag" sequence (such as a peptide) to facilitate purification. The marker or tag amino acid sequence can be a six-histidine peptide, a hemagglutinin ("HA") tag, and a "FLAG" tag.

[0269] Methods for fusing or conjugating moieties, including polypeptides, to antibodies are known (see, e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al., eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, Controlled Drug Delivery 623-53 (Robinson et al., eds., 2nd ed., 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al., eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy, Monoclonal Antibodies for Cancer Detection and Therapy 303-16 (Baldwin et al., ed., 1985); Thorpe et al., Immunol. Rev. 62:119-58 (1982); U.S. Pat. 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 307,434; EP 367,166; EP 394,827; PCT Publications 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. 154:5590-600 (1995); and Vil et al., Proc. Natl. Acad.Sci. USA 89:11337-41(1992)). .

[0270] Fusion proteins can be produced by, for example, gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling") techniques. DNA shuffling can be used to alter the activity of single-domain antibodies provided herein, including, for example, antibodies with higher affinity and lower dissociation rates (see, e.g., U.S. Patent 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 encoded antibodies can be altered by random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. The polynucleotides encoding the antibodies provided herein can be recombined with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

[0271] The single domain antibodies (eg, VHH domains) provided herein can be conjugated to a second antibody to form an antibody heteroconjugate.

[0272] In various embodiments, the single domain antibody is genetically fused to the agent. Genetic fusion can be achieved by placing a linker (e.g., a polypeptide) between the single domain antibody and the agent. The linker can be a flexible linker.

[0273] The single domain antibody can be genetically conjugated to a therapeutic molecule, wherein the hinge region connects the single domain antibody to the therapeutic molecule.

[0274] Also provided herein are methods for preparing the various fusion proteins provided herein. The various methods described in Section 5.2.6 above can also be used to prepare the fusion proteins provided herein.

[0275] In a specific embodiment, the fusion protein provided herein is recombinantly expressed. The recombinant expression of the fusion protein provided herein may require the construction of an expression vector containing a polynucleotide encoding the protein or its fragment. Once the polynucleotide encoding the protein provided herein or its fragment is obtained, the vector for producing the molecule can be produced by recombinant DNA technology using techniques well known in the art. Therefore, a method for preparing protein by expressing a polynucleotide containing a coding nucleotide sequence is described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing a coding sequence and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. A reproducible vector is also provided, which comprises a nucleotide sequence of a fusion protein or its fragment or CDR operably connected to a promoter.

[0276] The expression vector can be transferred to a host cell by conventional techniques, and then the transfected cells are cultured by conventional techniques to produce the fusion protein provided herein. Therefore, the present invention also provides a host cell containing a polynucleotide encoding a fusion protein or a fragment thereof provided herein that is operably linked to a heterologous promoter.

[0277] A variety of host expression vector systems can be used to express the fusion proteins provided herein. Such host expression systems represent vehicles, and the target coding sequence can be produced and subsequently purified by these vehicles, but also represent cells, and when transformed or transfected with the appropriate nucleotide coding sequence, these cells can express the fusion proteins provided herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence; yeast (e.g., Pichia pastoris) transformed with recombinant yeast expression vectors containing the coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence; plant cell systems infected with recombinant viral 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 systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) containing recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) 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, particularly bacterial cells for expressing complete recombinant antibody molecules, can be used to express recombinant fusion proteins. For example, mammalian cells such as Chinese hamster ovary cells (CHO) are combined with carriers such as the major intermediate early gene promoter elements from human cytomegalovirus to be an effective expression system for antibodies or their variants. In a specific embodiment, the expression of the nucleotide sequence encoding the fusion protein provided herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0278] In bacterial systems, various expression vectors can be advantageously selected according to the intended use of the expressed fusion protein. For example, when a large amount of such fusion proteins are to be produced, in order to produce a pharmaceutical composition of the fusion protein, it may be necessary to guide the expression of a high-level fusion protein product that is easy to purify. These vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 12: 1791 (1983)), in which the coding sequence can be linked to the vector alone in the frame with the lac Z coding region to produce a fusion protein; 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 exogenous polypeptides as fusion proteins with glutathione 5-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

[0279] In mammalian host cells, many virus-based expression systems can be used. In the case where adenovirus is used as an expression vector, the target coding sequence can be connected to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will produce a recombinant virus that is alive and can express fusion protein in an infected host (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1: 355-359 (1984)). The effective translation of the inserted coding sequence may also require a specific start signal. These signals include an ATG start codon and adjacent sequences. In addition, the start 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 start codons can have a variety of sources, including natural and synthetic. The efficiency of expression can be increased by including appropriate transcription enhancer elements, transcription terminators, and the like (see, eg, Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0280] In addition, host cell strains that regulate the expression of inserted sequences or modify and process gene products in a desired ad hoc manner can be selected. Such modifications (e.g., glycosylation) and processing (e.g., cutting) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign proteins. For this reason, eukaryotic host cells with cell mechanisms for appropriately processing primary transcripts, glycosylation, and gene product phosphorylation 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, BT20 and T47D, NS0 (mouse myeloma cell line that does not endogenously produce any immunoglobulin chain), CRL7030 and HsS78Bst cells.

[0281] In order to produce recombinant proteins for a long time and in high yield, stable expression can be utilized. For example, the cell line of the fusion protein can be stably expressed by engineering. Except using the expression vector containing the viral replication origin, the host cell can be transformed with the DNA controlled by suitable expression control elements (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and selective markers. After introducing foreign DNA, the engineered cells can be grown in enriched medium for 1-2 days and then transferred to selective medium. The selective marker in the recombinant plasmid confers resistance to selection, and allows the cell to stably integrate the plasmid into their chromosome and grow to form a focus, which can be cloned and expanded into a cell line. The method can be advantageously used for the cell line of the fusion protein expressed by engineering. This engineered cell line may be particularly useful in screening and evaluating the compositions that interact directly or indirectly with the binding molecule.

[0282] A number of 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 phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8-17 (1980)) genes can be used for tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection for 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):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology can be routinely used to select the 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), which are incorporated herein by reference in their entirety.

[0283] The expression level of fusion protein can be increased by vector amplification (for review, 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)). When the marker in the vector system expressing the fusion protein is amplifiable, the increase in the inhibitor level present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the fusion protein gene, the production of the fusion protein will also increase (Crouse et al., Mol. Cell. Biol. 3: 257 (1983)).

[0284] Host cells can be co-transfected with a variety of expression vectors provided herein. The vectors can contain identical selectable markers that can make the corresponding encoded polypeptides equally expressed. Alternatively, a single vector encoding and capable of expressing a variety of polypeptides can be used. The coding sequence can comprise cDNA or genomic DNA.

[0285] Once the fusion proteins provided herein have been produced by recombinant expression, they can be purified by any method known in the art for purifying polypeptides (e.g., immunoglobulin molecules), for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens after protein A, size column chromatography, and Kappa select affinity chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the fusion protein molecules provided herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0286] Immunoconjugates

[0287] The present disclosure also provides immunoconjugates comprising any of the antibodies described herein (such as an anti-claudin 6 single domain antibody) conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

[0288] Immunoconjugates can be antibody-drug conjugates (ADCs) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425235 B1); auristatins, such as 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); caudatelin; 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 Cell Biology, 2007; 15(5):145-156. Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as 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, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0289] The immunoconjugate may comprise an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modicin A chain, alpha-sarcin, Aleurites fordii proteins, Dianthus caryophyllus proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, toxin protein, crotonin, saponin inhibitor, gelonin, mitomycin, restrictocin, phenomycin, enomycin and telosporin.

[0290] Immunoconjugates may comprise an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to prepare radioconjugates. Examples include At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0291] 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 (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94 / 11026.

[0292] The linker can be a "cleavable linker" that facilitates release of the conjugate in the cell, but non-cleavable linkers are also contemplated herein. Linkers for use 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 comprising amino acids such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to circumvent multidrug transporter-mediated resistance.

[0293] The immunoconjugates or ADCs herein contemplate, but are not limited to, such conjugates prepared with cross-linker reagents including, but 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, and sulfo-SMPB and SVSB (succinimidyl-(4-vinyl sulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0294] In other embodiments, the antibodies provided herein are conjugated or recombinantly fused with, for example, diagnostic molecules. Such diagnosis and detection can be accomplished, for example, by coupling the antibody to a detectable substance, which includes, but is not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin or avidin / biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent materials, such as 225Acγ-emitting, Auger-emitting, β-emitting, α-emitting, or positron-emitting radioisotopes.

[0295] 5.3. Chimeric Antigen Receptors

[0296] On the other hand, a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain is provided herein, the extracellular antigen binding domain comprising a single domain antibody (e.g., VHH) provided herein that binds to tight junction protein 6. An exemplary CAR comprising a VHH domain of the present invention (i.e., a VHH-based CAR) is described in Section 6 below.

[0297] In some embodiments, the chimeric antigen receptor (CAR) provided herein comprises a polypeptide comprising: (a) an extracellular antigen binding domain comprising a single domain antibody (sdAb) specifically binding to tight junction protein 6 as provided herein, and optionally one or more additional binding domains; (b) a transmembrane domain; and (c) an intracellular signaling domain. Each component and additional region is described in more detail below.

[0298] 5.3.1. Extracellular antigen binding domain

[0299] The extracellular antigen binding domain of CAR described herein comprises one or more (such as any one of 1, 2, 3, 4, 5, 6 or more) single domain antibodies. Single domain antibodies can be directly fused to each other via a peptide bond or via a peptide linker.

[0300] The CAR disclosed herein comprises an extracellular antigen binding domain, which comprises one or more single domain antibodies. sdAb can have the same or different sources and have the same or different sizes. In some embodiments, the extracellular antigen binding domain provided herein comprises at least one binding domain, and the at least one binding domain comprises a single domain antibody that binds to tight junction protein 6 as provided herein, for example, an anti-tight junction protein 6 single domain antibody described in Section 5.2 above.

[0301] In some embodiments, provided herein is a CAR comprising a 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 an anti-claudin 6 sdAb as described in Section 5.2 above.

[0302] In some embodiments, provided herein is a CAR comprising a 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 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 7; (ii) CDR1, CDR2, and CDR3 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 8; (iii) CDR1, CDR2, and CDR3 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 9; (iv) CDR1, CDR2, and CDR3 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 10; or (v) CDR1, CDR2, and CDR3 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 11; NO: 11. In some embodiments, CDR1, CDR2, and CDR3 are 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) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5; or (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and a 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 to the sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0303] In other embodiments, the extracellular antigen binding domain comprises two or more antigen binding domains. Of the two or more antigen binding domains, at least one is a VHH that binds to tight junction protein 6 as provided herein, and one or more additional binding domains that bind to one or more additional antigens, for example, 1, 2, 3, 4 or more additional single domain antibody binding regions (sdAb) 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 domains comprise HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 18 and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 19. In some embodiments, CDR1, CDR2 or CDR3 are 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 a HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, and a LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the additional binding domain comprises a VH domain comprising SEQ ID NO: 18 and a VL domain comprising 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.

[0304] In addition to one or more antigen binding domains provided herein, the CAR provided herein may further comprise one or more of the following: 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, each of which is described in more detail below.

[0305] For example, in some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as 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 a group consisting of a ligand of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and a combination thereof. In some embodiments, the co-stimulatory signaling domain is derived from CD137. In some embodiments, the tight junction protein 6 CAR further comprises a hinge domain (such as 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 tight junction protein 6 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from N-terminus to 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 tight junction protein 6 CAR is monospecific. In some embodiments, the tight junction protein 6 CAR is monovalent. In some embodiments, the tight junction protein 6×GPC3 CAR provided herein is bispecific.

[0306] Peptide linker

[0307] In the case where there are multiple antibodies (e.g., multiple antibody fragments) in the CAR of the present invention, the various antibodies can be fused to each other via a peptide linker. Antibodies can be directly fused to each other in the absence of any peptide linker. The peptide linkers connecting different antibodies can be the same or different. The different domains of CAR can also be fused to each other via a peptide linker.

[0308] Depending on the structure and / or functional characteristics of the antibody and / or various domains, each peptide linker in CAR may have the same or different lengths and / or sequences. Each peptide linker may be selected and optimized independently. The length, flexibility and / or other properties of one or more peptide linkers used in CAR may have some effects on properties, including but not limited to 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 interfere with each other spatially. A short peptide linker may be arranged between the transmembrane domain and the intracellular signaling domain of CAR. A peptide linker may include flexible residues (such as glycine and serine) so that adjacent domains are free to move relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker.

[0309] The peptide linker can have any suitable length. The length of the peptide linker can be at least about any one of the following: 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 can be no more than about any one of the following: 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less amino acids. The length of the peptide linker can be any of the following: 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 long, 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.

[0310] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO 1996 / 34103. The peptide linker may be a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G) n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers and other flexible joints known in the art. Other joints known in the art, for example, as WO2016014789, WO 2015158671, WO 2016102965, US 20150299317, WO 2018067992, US7741465, Colcher et al., J.Nat.Cancer Inst.82:1191-1197 (1990) and Bird et al., Science242:423-426 (1988) described in, may also be included in CAR provided herein, and their respective disclosures are incorporated herein by reference.

[0311] In some specific embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41.

[0312] 5.3.2. Transmembrane domain

[0313] The CAR of the present disclosure comprises a transmembrane domain that can be fused directly or indirectly to an extracellular antigen binding domain. The transmembrane domain can be derived from a natural or synthetic source. As used herein, a "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, such as a eukaryotic cell membrane. The transmembrane domain suitable for the CAR described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, for example, a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0314] According to the three-dimensional structure of the transmembrane domain, the transmembrane domain is classified.For example, the transmembrane domain can form an alpha spiral, a complex of more than one alpha spiral, a beta barrel or any other stable structure that can cross the phospholipid bilayer of the cell.In addition, the transmembrane domain can also or alternatively be classified according to the transmembrane domain topology, including the number of times the transmembrane domain passes through the membrane and the orientation of the protein.For example, a single transmembrane protein passes through the cell membrane once, and multiple transmembrane proteins pass through the cell membrane at least twice (for example, 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 topological structure of its end and one or more membranes through the section relative to the inside and outside of the cell.Type I membrane proteins have a single transmembrane region, and it is oriented so that the N-terminal of the protein is present in the cell extracellular of the lipid bilayer of the cell, and the C-terminal of the protein is present in the intracellular side.Type II membrane proteins also have a single transmembrane region, but it is oriented so that the C-terminal of the protein is present in the cell extracellular of the lipid bilayer of the cell, and the N-terminal of the protein is present in the intracellular side. Type III membrane proteins have multiple transmembrane segments and can be further classified according to the number of transmembrane segments and the locations of the N-termini and C-termini.

[0315] The transmembrane domain of CAR described herein can be derived from type I single transmembrane protein.The transmembrane domain from multi-transmembrane protein can also be compatible with CAR described herein.Multi-transmembrane protein can include complex (at least 2,3,4,5,6,7 or more) alpha helices or beta folded structures.The N-terminus and C-terminus of multi-transmembrane protein can be present on the opposite side of lipid bilayer, for example, the N-terminus of protein is present on the intracellular side of lipid bilayer, and the C-terminus of the protein is present on the extracellular side.

[0316] The transmembrane domain of CAR may comprise a transmembrane domain selected from the group consisting of: α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, tight junction protein 6, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, the transmembrane domain of 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.

[0317] In some specific embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain is a transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO:36.

[0318] The transmembrane domain for CAR described herein can also include at least a portion of a synthetic, non-naturally occurring protein segment. The transmembrane domain can be a synthetic, non-naturally occurring alpha helix or beta fold. The protein segment can 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, for example, in U.S. Patent No. 7,052,906 and PCT Publication No. WO2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0319] The membrane-spanning domain provided herein can include a membrane-spanning region and an intracellular region located at the C-terminal side of the membrane-spanning domain. The intracellular region of the membrane-spanning domain can include three or more amino acids, and in some embodiments, helps to orient the membrane-spanning domain in a lipid bilayer. In certain embodiments, one or more cysteine ​​residues are present in the membrane-spanning region of the membrane-spanning domain. One or more cysteine ​​residues may be present in the intracellular region of the membrane-spanning domain. The intracellular region of the membrane-spanning domain may include positively charged amino acids. The intracellular region of the membrane-spanning domain may include amino acids arginine, serine and lysine.

[0320] The membrane-spanning region of the membrane-spanning domain may include hydrophobic amino acid residues. The membrane-spanning domain of the CAR provided herein may include an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan and valine may be present at the C-terminus of the membrane-spanning domain. The membrane-spanning region may mainly include hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine. The membrane-spanning region may be hydrophobic. The membrane-spanning region may include a polyleucine-alanine sequence. The hydrophilicity or hydrophobicity or hydrophilicity characteristics of a protein or protein segment may be assessed by any method known in the art, such as Kyte and Doolittle hydrophilicity analysis.

[0321] 5.3.3. Intracellular signaling domain

[0322] The CAR of the present disclosure comprises an intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one normal effector function of the immune effector cell expressing the CAR. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity, or auxiliary activity, including secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a protein portion that transduces effector function signals and instructs cells to perform specific functions. Although the entire intracellular signaling domain can usually be used, in many cases the entire chain does not need to be used. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of a complete chain, as long as it transduces an effector function signal. Therefore, the term intracellular signaling domain means any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0323] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. "Primary intracellular signaling domain" refers to an intracellular signaling sequence that acts in a stimulatory manner to induce immune effector function. The primary intracellular signaling domain may contain a signaling motif called an activation motif based on immunoreceptor tyrosine or ITAM. As used herein, "ITAM" is a conserved protein motif that is typically present in the tail of a signaling molecule expressed in many immune cells. The motif may comprise two repeats of an amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing a conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in ITAMs after activation of signaling molecules. ITAMs may also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary intracellular signaling sequences include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0324] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. The intracellular signaling domain may consist 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.

[0325] 5.3.4. Co-stimulatory signaling domain

[0326] In addition to stimulating antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, and to activate the effector functions of cells. In some embodiments, 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 signal transduction 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 that transduces signals and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils."Co-stimulatory signaling domain" may be an intracellular portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognate binding partner on an immune cell (e.g., T cell) that specifically binds to a co-stimulatory ligand to mediate a co-stimulatory response of an immune cell, such as, but not limited to, proliferation and survival.

[0327] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (e.g., about any one of 2, 3, 4 or more) costimulatory signaling domains. The intracellular signaling domain may comprise two or more identical costimulatory signaling domains. The intracellular signaling domain may comprise two or more costimulatory signaling domains from different costimulatory proteins (such as any two or more costimulatory proteins described herein). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as an intracellular signaling domain of CD3ζ) and one or more costimulatory signaling domains. One or more costimulatory signaling domains and the primary intracellular signaling domain (such as the intracellular signaling domain of CD3ζ) may be fused to each other via an optional peptide linker. The primary intracellular signaling domain and the one or more costimulatory signaling domains may be arranged in any suitable order. In some embodiments, one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the intracellular signaling domain of CD3ζ). Multiple co-stimulatory signaling domains can provide additive or synergistic stimulation.

[0328] The activation of the costimulatory signaling domain in the host cell (e.g., immune cell) can induce cells to increase or reduce the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be applicable to CAR as described herein. One or more types of costimulatory signaling domains are based on factors such as the type of immune effector cells (e.g., T cells, NK cells, macrophages, neutrophils or eosinophils) and the desired immune effector functions (e.g., ADCC effects) in which the effector molecules are expressed and selected.Examples of costimulatory signaling domains for CAR can be intracellular signaling domains of costimulatory proteins, including but not limited to 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); 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 TNF RII / 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 co-stimulatory molecules, such as CD2, CD7, CD53, CD82 / K ai-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.

[0329] In some embodiments, the one or more co-stimulatory signaling domains are selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and combinations thereof.

[0330] In some embodiments, the intracellular signaling domain in the CAR of the present disclosure comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of CD3 ζ and a costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain of CD137 comprising an amino acid sequence of SEQ ID NO: 37.

[0331] Variants of any co-stimulatory signaling domain described herein are also within the scope of the present disclosure, so that the co-stimulatory signaling domain can regulate the immune response of immune cells. Compared to the wild-type control, the co-stimulatory signaling domain can include up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5 or 8). Such co-stimulatory signaling domains comprising one or more amino acid changes can be referred to as variants. Relative to the co-stimulatory signaling domain that does not contain a mutation, the mutation of the amino acid residues of the co-stimulatory signaling domain can lead to an increase in signal transduction and enhanced stimulation of the immune response. Relative to the co-stimulatory signaling domain that does not contain a mutation, the mutation of the amino acid residues of the co-stimulatory signaling domain can lead to a decrease in signal transduction and reduced stimulation of the immune response.

[0332] 5.3.5. Hinge region

[0333] The CAR of the present disclosure may include a hinge domain between an extracellular antigen binding domain and a transmembrane domain. The hinge domain is an amino acid segment usually found between two domains of a protein, and can allow the flexibility of the protein and the movement of one or two domains relative to each other. Any amino acid sequence that provides this flexibility and movement of the transmembrane domain of an effector molecule with respect to an extracellular antigen binding domain can be used.

[0334] The hinge domain can contain about 10-100 amino acids, for example, about 15-75 amino acids, 20-50 amino acids, or any of 30-60 amino acids. The hinge domain can be at least 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 in length.

[0335] The hinge domain can be a hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art comprising a hinge domain is suitable for chimeric receptors described herein. The hinge domain can be at least a portion of the hinge domain of a naturally occurring protein, and imparts flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. The hinge domain can be a part of the hinge domain of CD8α, for example, a fragment of at least 15 (e.g., 20, 25, 30, 35 or 40) continuous amino acids of the hinge domain containing CD8α. In some embodiments, the hinge domain of CD8α comprises the amino acid sequence of SEQ ID NO:35.

[0336] Hinge domains of antibodies (such as IgG, IgA, IgM, IgE or IgD antibodies) are also suitable for pH-dependent chimeric receptor systems described herein. The hinge domain can be a hinge domain that connects the constant domains CH1 and CH2 of the antibody. The hinge domain can be a hinge domain of an antibody, and comprises a hinge domain of one or more constant regions of the antibody and the antibody. The hinge domain can comprise a hinge domain of the CH3 constant region of the antibody and the antibody. The hinge domain can comprise a hinge domain of the CH2 and CH3 constant regions of the antibody and the antibody. The antibody can be an IgG, IgA, IgM, IgE or IgD antibody. Optionally, the antibody is an IgG antibody. The antibody can be an IgG1, IgG2, IgG3 or IgG4 antibody. The hinge region can comprise the hinge region of an IgG1 antibody and CH2 and CH3 constant regions. The hinge region can comprise the hinge region and CH3 constant region of an IgG1 antibody.

[0337] Non-naturally occurring peptides can also be used as hinge domains 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 can be a peptide linker, such as a (GxS)n linker, wherein x and n can independently be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.

[0338] 5.3.6. Signal peptide

[0339] The CAR of the present disclosure may include a signal peptide (also referred to as a signal sequence) at the N-terminus of a polypeptide. Typically, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. The signal peptide can target the secretory pathway of the effector molecule to the cell and allows the effector molecule to be integrated and anchored into a lipid bilayer. It will be apparent to those skilled in the art that the signal peptides suitable for use in CAR described herein and comprising a signal sequence of a naturally occurring protein or a synthetic, non-naturally occurring signal sequence. The signal peptide may be derived from a molecule selected from a group consisting of CD8 α, GM-CSF receptor α, and IgG1 heavy chains. In some embodiments, the signal peptide is derived from CD8 α. In some embodiments, the signal peptide of CD8 α includes the amino acid sequence of SEQ ID NO:34.

[0340] 5.3.7. Exemplary CARs that bind to Claudin 6 and / or GPC3

[0341] An exemplary CAR that binds to tight junction protein 6 is generated as shown in Section 6 below, such as CNBCAR1. In some embodiments, a CAR comprising an amino acid sequence of SEQ ID NO: 26 or consisting of an amino acid sequence of SEQ ID NO: 26 is provided herein. In certain embodiments, the CAR provided herein comprises an amino acid sequence having a certain percentage of identity with respect to any of the CARs exemplified in Section 6 below. In some embodiments, a tight junction protein 6 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 SEQID NO: 26 is provided herein.

[0342] In some embodiments, the present invention provides a nucleic acid encoding any tight junction protein 6 CAR provided herein. A more detailed description of nucleic acid sequences and vectors is provided below.

[0343] Exemplary CARs that bind to tight junction protein 6 and GPC3 are generated as shown in Section 6 below, such as CNBCAR3. In some embodiments, a CAR comprising an amino acid sequence of SEQ ID NO: 28 or consisting of an amino acid sequence of SEQ ID NO: 28 is provided herein. In certain embodiments, the CAR provided herein comprises an amino acid sequence having a certain percentage of identity relative to any of the CARs exemplified in Section 6 below. In some embodiments, a tight junction protein 6×GPC3CAR 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 is provided herein.

[0344] In some embodiments, the present invention provides a nucleic acid encoding any tight junction protein 6×GPC3CAR provided herein. A more detailed description of nucleic acid sequences and vectors is provided below.

[0345] Other exemplary CARs provided herein further comprise chimeric receptors. In some embodiments, the chimeric receptor comprises TGFβR and / or IL23R. The CAR provided herein may comprise the amino acid sequence of SEQ ID NO:29. The CAR provided herein may comprise the amino acid sequence of SEQ ID NO:31. 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 to the amino acid sequence of SEQ ID NO:29 may be provided herein. The present invention may 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 to the amino acid sequence of SEQ ID NO:31.

[0346] In certain embodiments, a nucleic acid encoding any CAR and chimeric receptor provided herein is provided herein. A more detailed description of nucleic acid sequences and vectors is provided below.

[0347] 5.4. Engineered immune effector cells

[0348] In other aspects, provided herein are host cells (such as immune effector cells) comprising any of the CARs described herein.

[0349] Thus, in some embodiments, provided herein is an engineered immune effector cell (such as a T cell) comprising a CAR, the CAR comprising a 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 an anti-claudin 6 sdAb as described in Section 5.2 above, including, for example, those comprising: (i) CDR1, CDR2, and CDR3 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 7; (ii) CDR1, CDR2, and CDR3 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 8; (iii) CDR1, CDR2, and CDR3 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 9; (iv) CDR1, CDR2, and CDR3 having amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 10; NO:10; or (v) having CDR1, CDR2 and CDR3 of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO:11, respectively. 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) a CDR1 comprising the amino acid sequence of SEQ ID NO:1; a CDR2 comprising the amino acid sequence of SEQ ID NO:3; and a CDR3 comprising the amino acid sequence of SEQ ID NO:5; or (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:4; and a 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 to the sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0350] In some embodiments, the extracellular antigen binding domain further comprises one or more additional antigen binding domains. In some embodiments, the additional binding domain comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 18 and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 19. In some embodiments, CDR1, CDR2 or CDR3 are 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 a HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, as well as a LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the additional binding domain comprises a VH domain comprising SEQ ID NO: 18 and a VL domain comprising 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. In some embodiments, the peptide linker is no more than about 50 amino acids long. 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 a primary intracellular signaling domain of an immune effector cell (such as 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 a group consisting of a ligand of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and a combination thereof. In some embodiments, CAR further comprises a hinge domain (such as 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, CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus: a CD8α signal peptide, an extracellular antigen binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ.

[0351] In other specific embodiments, provided herein is an engineered immune effector cell (such as a T cell) comprising a CAR, the CAR comprising a 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.

[0352] In some embodiments, the engineered immune effector cell is a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, or an embryonic stem cell. The T cell can be an αβT cell or a γδT cell. The engineered immune effector cell can be autologous. The engineered immune effector cell can be heterologous.

[0353] The engineered immune effector cells can further express one or more therapeutic proteins and / or immunomodulators, such as immune checkpoint inhibitors.

[0354] 5.4.2. Carrier

[0355] The present disclosure provides a vector for cloning and expressing any of the CARs described herein. The vector is suitable for replication and integration in eukaryotic cells such as mammalian cells. The vector can 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 virus vectors and derivatives thereof. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0356] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or in vitro. Many retroviral systems are known in the art. Adenoviral vectors can be used. Many adenoviral vectors are known in the art. Slow virus vectors can be used. Self-inactivating slow virus vectors can be used. For example, self-inactivating slow virus vectors carrying immunomodulators (such as immune checkpoint inhibitors) coding sequences and / or self-inactivating slow virus vectors carrying chimeric antigen receptors can be packaged with schemes known in the art. Methods known in the art can be used to transduce mammalian cells (e.g., primary human T cells). Vectors derived from retroviruses such as slow viruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenics and their propagation in daughter cells. Slow virus vectors also have low immunogenicity and can transduce non-proliferating cells.

[0357] In some embodiments, the vector comprises any one of the nucleic acids encoding CAR described herein. The nucleic acid can be cloned into a vector using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selective markers. The nucleic acid can be operably connected to a promoter. A variety of promoters for gene expression in mammalian cells have been studied, and any promoter known in the art can be used for the present disclosure. Promoters can be roughly divided into constitutive promoters or regulated promoters, such as inducible promoters.

[0358] The nucleic acid encoding CAR can be operably connected to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters considered herein include, but are not limited to, cytomegalovirus (CMV) promoters, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter (CAGG) coupled to CMV early enhancer. In many studies, such constitutive promoters have been widely compared for the efficiency of driving transgenic expression. For example, Michael C.Milone et al. compared the efficiency of CMV, hEF1α, UbiC and PGK driving chimeric antigen receptor expression in primary human T cells, and concluded that hEF1α promoters not only induced the highest level of transgenic expression, but also optimally maintained in CD4 and CD8 human T cells (Molecular Therapy, 17 (8): 1453-1464 (2009)). The nucleic acid encoding CAR can be operably linked to a hEF1α promoter.

[0359] The nucleic acid encoding CAR can be operably connected to an inducible promoter. Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of engineered immune effector cells or the physiological state of engineered immune effector cells, inducers (ie, inducers) or combinations thereof.

[0360] The inducing conditions may not induce expression of endogenous genes in the engineered mammalian cells and / or the subject receiving the pharmaceutical composition. The inducing conditions may be selected from the group consisting of: an inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and activation state of the engineered mammalian cells.

[0361] The vector may also contain a selective marker gene or a reporter gene to select cells expressing CAR from a host cell population transfected by a lentiviral vector. Both the selective marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of nucleic acid sequences.

[0362] The vector may include more than one nucleic acid encoding CAR. The vector may include 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 operably connected 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.

[0363] 5.4.3. Immune effector cells

[0364] "Immune effector cell" refers to an immune cell that can exert an immune effector function. An immune effector cell can express at least FcγRIII and exert an ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0365] In some embodiments, immune effector cells are T cells. T cells can be αβT cells or γδT cells. T cells can be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8- or a combination thereof. T cells can produce IL-2, TFN and / or TNF after expressing CAR and binding to target cells (such as GPC3+ tumor cells). CD8+T cells can lyse antigen-specific target cells after expressing CAR and binding to target cells.

[0366] In some embodiments, the immune effector cells are NK cells. The immune effector cells can be established cell lines, such as NK-92 cells.

[0367] In some embodiments, the immune effector cells are differentiated from stem cells (eg, hematopoietic stem cells, pluripotent stem cells, iPS cells, or embryonic stem cells).

[0368] Engineered immune effector cells are prepared by introducing CAR into immune effector cells such as T cells. In some embodiments, CAR is introduced into immune effector cells by transfection of any nucleic acid or any of the above vectors. By inserting the protein into the cell membrane while passing the cell through a microfluidic system (such as CELL ), CAR can be introduced into immune effector cells (see, e.g., U.S. Patent Application Publication No. 20140287509).

[0369] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical or biological methods.

[0370] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. 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. Vectors can be introduced into cells by electroporation.

[0371] Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, such as human cells.

[0372] Chemical methods for introducing carriers into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0373] The RNA molecules encoding any CAR described herein can be prepared by conventional methods (e.g., in vitro transcription), and then introduced into immune effector cells by known methods such as mRNA electroporation. See, for example, Rabinovich et al., Human Gene Therapy 17: 1027-1035 (2006).

[0374] In certain embodiments, the transduced or transfected immune effector cells are propagated in vitro after the introduction of a vector or nucleic acid. The transduced or transfected immune effector cells can be cultured to breed at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days or 14 days. The transduced or transfected immune effector cells can be further evaluated or screened to select engineered mammalian cells.

[0375] Reporter gene can be used to identify cells that may be transfected and to evaluate the function of regulatory sequences. Usually, reporter gene is not present in or is not expressed by receptor organisms or tissues and encodes a gene that is expressed as a polypeptide by some easily detectable properties (e.g., enzymatic activity). The expression of reporter gene is detected at the appropriate time after DNA is introduced into the receptor cells. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (e.g., Ui-Tei et al. FEBS Letters 479: 79-82 (2000)). Suitable expression system is well known and can be prepared using known techniques or commercially available.

[0376] Other methods for confirming the presence of CAR-encoding nucleic acids in engineered immune effector cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of specific peptides by immunological methods such as ELISA and Western blotting.

[0377] 5.4.4. T cell sources

[0378] Before T cells are amplified and genetically modified, T cell sources can be obtained from subjects. 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 infection site, ascites, pleural effusion, spleen tissue and tumor. Any number of T cell lines available in the art can be used. Various techniques known to technicians (such as Ficoll) can be used. TM T cells are obtained from a unit of blood collected from a subject by separation. Cells from individual circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. Cells collected by apheresis can be washed to remove the plasma portion, and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. The cells are washed with phosphate buffered saline (PBS). The washing solution lacks calcium and may lack magnesium or may lack many (if not all) divalent cations. The initial activation step in the absence of calcium can lead to amplified activation. It is readily understood by those of ordinary skill in the art that the washing step can be completed by methods known in the art, such as using a semi-automated "flow" 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-free 2+ , Mg-free 2+ Alternatively, undesirable components of the aliquot can be removed and the cells resuspended directly in culture medium.

[0379] This can be done, for example, by PERCOLL TM T cells are separated from peripheral blood lymphocytes by gradient centrifugation or by countercurrent centrifugal elutriation to lyse red blood cells and deplete monocytes. 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, by conjugating beads (such as anti-CD3 / anti-CD28 (i.e., 3×28) to M-450 CD3 / CD28 T) is incubated for a period of time sufficient to positively select the desired T cells and separate T cells. The time period can be about 30 minutes. The range of the time period can be 30 minutes to 36 hours or longer, and all integer values ​​therebetween. The time period can be at least 1, 2, 3, 4, 5 or 6 hours. The time period can be 10 to 24 hours. The incubation period can be 24 hours. For the separation of T cells of leukemia patients, using a longer incubation time (such as 24 hours) can increase cell yield. Compared with other cell types, in any case where T cells are less, such as in the case of separating tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals, a longer incubation time can be used to separate T cells. In addition, using a longer incubation time can increase the capture efficiency of CD8+T cells. Therefore, in some embodiments, by simply shortening or extending the time allowing T cells to bind to CD3 / CD28 beads and / or increasing or decreasing the ratio of beads to T cells, a subpopulation of T cells can be preferentially selected or not selected at the beginning of culture or at other time points during culture. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, a subset of T cells may be preferentially selected or not selected at the start of the culture or at other desired time points. One skilled in the art will recognize that multiple rounds of selection may also be used. It may be desirable to perform a selection procedure and use "unselected" cells during activation and amplification. "Unselected" cells may also be subjected to further rounds of selection.

[0380] Enrichment of T cell populations by negative selection can be accomplished by binding antibodies against surface markers unique to the negatively selected cells. One approach is to perform cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich for CD4 + Cells, the monoclonal antibody cocktail 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, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0381] In order to separate the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles, such as beads) can be changed. It may be desirable to significantly reduce the volume of beads and cells mixed together (i.e., increase the concentration of cells) to ensure maximum contact between cells and beads. For example, a concentration of 2 billion cells / ml is used. A concentration of 1 billion cells / ml can be used. Greater than 100 million cells / ml can be used. A concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml can be used. A concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml can be used. A concentration of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, the use of high concentrations of cells can allow for more efficient capture of cells that may weakly express the target antigen of interest (such as CD28 negative T cells), or cells from samples where there are many tumor cells (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are desirable to obtain. The use of high cell concentrations can allow for more efficient selection of CD8+ T cells that typically have weaker CD28 expression.

[0382] It may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between particles and cells are minimized. This selects for cells that express large amounts of the desired antigen that binds to the particles. For example, CD4+ T cells express higher levels of CD28 and are captured more efficiently than dilute concentrations of CD8+ T cells. The concentration of cells used may be 5×10 6 / mL. The concentration used can be about 1×10 5 / mL to 1×10 6 pieces / mL, and any integer value in between.

[0383] Cells can be incubated on a rotator at different speeds at 2°C-10°C or at room temperature for different lengths of time.

[0384] T cells for stimulation can also be frozen after the washing step. Without being bound by theory, freezing and subsequent thawing steps can provide a more uniform product by removing granulocytes in the cell population and to some extent monocytes. After the washing steps of removing plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and are useful in this case, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 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 culture medium, or containing other suitable cell freezing culture mediums such as Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1° / min and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0385] Cryopreserved cells can be thawed and washed as described herein and allowed to stand at room temperature for one hour prior to activation.

[0386] It is also contemplated in the present disclosure that a blood sample or a single sampling product is collected from a subject in a time period before the cells that may be amplified as described herein are needed. Therefore, the cell source to be amplified can be collected at any necessary time point, and the required cells, such as T cells, can be separated and frozen to be subsequently used in T cell therapy for various diseases or conditions that will benefit from T cell therapy, such as those described herein. A blood sample or a single sampling can be taken from a generally healthy subject. A blood sample or a single sampling of blood components can be taken from a generally healthy subject who is at risk of disease but not yet diseased, and the target cells are separated and frozen for later use. T cells can be amplified, frozen and used later. In certain embodiments, a sample is collected from a patient shortly after diagnosing a specific disease as described herein but before any treatment. Cells may be isolated from a blood sample or apheresis of a subject prior to various relevant treatment modalities including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablative agents (such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228), and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (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 frozen for subsequent use in combination with (e.g., before, simultaneously with, or after) bone marrow or stem cell transplantation, T cell depletion therapy using chemotherapeutic agents (such as fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or antibodies (such as OKT3 or CAMPATH).

[0387] T cells can be obtained directly from patients after treatment. In this regard, it has been observed that after some cancer treatments, particularly after treatment with drugs that damage the immune system, in the period when the patient usually recovers from treatment, soon after treatment, the quality of the T cells obtained may be optimal, or improve the ability of its ex vivo expansion. Similarly, after ex vivo operation using the method described herein, these cells can be in a preferred state of enhanced implantation and in vivo expansion. Therefore, it is expected in the context of the present disclosure that blood cells, including T cells, dendritic cells or other hematopoietic cells, are collected in this recovery phase. In addition, mobilization (for example, mobilization with GM-CSF) and conditioning schemes can be used to create conditions in a subject, wherein particularly during the time window defined after treatment, it is conducive to the refilling, recycling, regeneration and / or expansion of cell types. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0388] 5.4.5. T cell activation and expansion

[0389] In some embodiments, before or after genetic modification of T cells with a CAR described herein, the T cells can be activated and expanded generally using methods such as those described in the following documents: 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.

[0390] Typically, T cells can be expanded by contacting with the surface of the part of the co-stimulatory molecule on the surface of the reagent and the stimulatory molecule attached with the relevant signal of the CD3 / TCR complex. Specifically, T cell colonies can be stimulated as described herein, such as by contacting with anti-CD3 antibodies or their antigen-binding fragments or anti-CD2 antibodies fixed on the surface or by contacting with a protein kinase C activator (e.g., bryostatin) combined with a calcium ion carrier. In order to costimulate the auxiliary molecules on the surface of T cells, the part of the auxiliary molecule can be used. For example, under conditions suitable for stimulating T cell proliferation, T cell colonies can be contacted with anti-CD3 antibodies and anti-CD28 antibodies. In order to stimulate the proliferation of CD4+T cells or CD8+T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. Examples of anti-CD3 antibodies include UCHT1, OKT3, HIT3a (BioLegend, San Diego, US), which can be used as other methods known in the art (Graves J et al., J. Immunol. 146:2102 (1991); Li B, et al., Immunology 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), as well as other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, (1998); Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63 (1999)).

[0391] The primary stimulation signal and the costimulatory signal of T cell can be provided by different schemes.For example, the medicament providing each signal can be in solution or coupled to the surface.When coupled to the surface, the medicament can be coupled to the same surface (that is, in "cis" form) or to a separate surface (that is, in "trans" form).Alternatively, a kind of medicament can be coupled to the surface and another medicament in solution.The reagent providing the costimulatory signal can be bound to the cell surface and the reagent providing the primary activation signal can be in solution or coupled to the surface.Both medicaments can be in solution.The medicament can be in soluble form, then cross-linked to the surface, such as cells or antibodies expressing Fc receptors or other binding agents that will be combined with medicaments.In this regard, referring to, for example, the artificial antigen presenting cells (aAPC) of U.S. Patent Application Publication Nos. 20040101519 and 20060034810, they are considered to be used for activating and amplifying T cells in some embodiments of the present disclosure.

[0392] In some embodiments, T cells are combined with beads coated with reagents, beads and cells are subsequently separated, and cells are then cultured. Before culture, beads and cells coated with reagents may not be separated, but cultured together. Beads and cells may first be concentrated by applying a force (such as a magnetic force), resulting in increased connection of cell surface markers, thereby inducing cell stimulation.

[0393] For example, cell surface proteins can be linked by allowing paramagnetic beads (3×28 beads) attached with anti-CD3 and anti-CD28 to contact T cells. 4 Up to 4×10 8 T cells) and beads (e.g., anti-CD3 / CD28 MACSiBead particles with a recommended titer of 1:100) are combined in a buffer, such as PBS (without divalent cations, such as calcium and magnesium). It will be readily appreciated by one of ordinary skill in the art that any cell concentration may be used. For example, the target cells may be very few 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 number of cells is within the scope of the present disclosure. It may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of the cells) to ensure maximum contact between the cells and the particles. For example, a concentration of about 2 billion cells / mL may be used. Greater than 100 million cells / mL may be used. A concentration 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. A concentration of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / mL can be used. A concentration of 125 or 150 million cells / mL can be used. Using high concentrations can lead to increased cell yield, cell activation and cell expansion. In addition, using high cell concentrations can allow 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 in certain embodiments it is desirable to be able to obtain. For example, using high concentrations of cells allows more effective selection of CD8+T cells that generally have weaker CD28 expression.

[0394] The mixture can be cultured for several hours (about 3 hours) to about 14 days or any hour integer value therebetween. The mixture can be cultured for 21 days. The beads and T cells are cultured together for about eight days. The beads and T cells can be cultured together for 2-3 days. Several stimulation cycles may also be required so that the culture time of T cells can be 60 days or longer. Conditions suitable for T cell culture include appropriate culture medium (e.g., minimum essential medium or RPMI culture medium 1640 or X-vivo 15 (Lonza)), which culture medium may contain factors necessary for proliferation and vigor, including 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 known to those skilled in the art for cell growth. Other additives for cell growth include but are not limited to surfactants, human plasma protein powder and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture medium can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-vivo 15 and X-vivo 20, optimizer, added with amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of determined hormones, and / or cytokines sufficient to grow and expand T cells. Antibiotics (such as penicillin and streptomycin) are only included in experimental cultures, not in cell cultures to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to different stimulation times can exhibit different characteristics. For example, a typical peripheral blood mononuclear cell product of blood or single blood components has more helper T cell populations (TH, CD4+) than cytotoxic or suppressive T cell populations (TC, CD8). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a T cell population that is primarily composed of TH cells before about day 8-9, and after about day 8-9, the T cell population contains an increasing number of TC cells. Therefore, depending on the purpose of treatment, it may be advantageous to infuse a subject with a T cell population that primarily contains TH cells. Similarly, if an antigen-specific subset of TC cells is isolated, it may be beneficial to expand that subset to a greater extent.

[0395] Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers also differed significantly but were largely reproducible during cell expansion. Thus, this reproducibility enables customization of activated T cell products for specific purposes.

[0396] 5.4.6. CAR-T cells expressing exogenously introduced TGFβR and IL23R

[0397] In certain embodiments, the T cells provided herein further express an exogenously introduced chimeric receptor comprising TGFβR and IL23R (also referred to herein as "TF23").

[0398] More specifically, in some embodiments, CAR-T cells expressing exogenously introduced TGFβR and IL23R can be generated by introducing one or more nucleic acids encoding polypeptides comprising both TGFβR and IL23R.

[0399] CAR, TGFβR and IL23R can each be introduced into T cells as separate polypeptides. For example, nucleic acids encoding CAR provided herein, nucleic acids encoding TGFβR and nucleic acids encoding IL23R are introduced into T cells separately.

[0400] Alternatively, any two of the three or all three of them can be introduced into T cells as a single polypeptide together by a nucleic acid, which will be cut when translated in the cell. For example, a nucleic acid encoding a polypeptide comprising a CAR provided herein and a TGFβR connected via a self-cleaving peptide linker is introduced into a T cell, and a nucleic acid encoding IL23R is introduced into a T cell separately. Similarly, a nucleic acid encoding a polypeptide comprising a CAR provided herein and an IL23R connected via a self-cleaving peptide linker is introduced into a T cell, and a nucleic acid encoding TGFβR is introduced into a T cell separately. In some embodiments, nucleic acids encoding polypeptides of all three of CAR, TGFβR and IL23R connected to each other by a self-cleaving peptide linker can be introduced into T cells. The self-cleaving peptide linker is described in more detail above. The 2A self-cleaving peptide may be selected from a group consisting of F2A, E2A, P2A, T2A or its variants. The self-cleaving peptide may be a 2A self-cleaving peptide P2A fragment comprising an amino acid sequence of SEQ ID NO:42.

[0401] Alternatively, the CAR-T cells provided herein can be produced by polynucleotides comprising multiple regions, such as regions encoding CAR, regions encoding TGFβR, and / or regions encoding IL23R. Different regions can be controlled by the same promoter. For example, in some embodiments, an internal ribosome entry site (IRES) is used herein to express multiple genes from a promoter. In other embodiments, different regions are controlled by separate promoters.

[0402] In some specific embodiments, the CAR-T cells provided herein are exogenously introduced with a TF23 chimeric receptor, which TF23 chimeric receptor comprises: a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of IL-12Rβ1, a first intracellular domain comprising the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of IL-23R, and a second intracellular domain comprising the intracellular domain of IL-23R, as described above. Fig. 7A In some specific embodiments, the CAR-T cells provided herein express an exogenously introduced polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 and 44-45. In other specific embodiments, the CAR-T cells provided herein exogenously introduce a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:32, such as a polynucleotide comprising a nucleic acid sequence of SEQ ID NO:33.

[0403] 5.5. Polynucleotides

[0404] In certain embodiments, the present disclosure provides polynucleotides encoding antibodies of the present invention (e.g., VHH domain antibodies) that bind to tight junction protein 6 and fusion proteins comprising antibodies that bind to tight junction protein 6 described herein. The polynucleotides disclosed herein can be in RNA form or DNA form. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding (antisense) strand. The polynucleotide can be in the form of cDNA. The polynucleotide can be a synthetic polynucleotide.

[0405] In certain embodiments, the disclosure provides polynucleotides encoding tight junction protein 6 binding CAR provided herein. The polynucleotides disclosed herein can be in RNA form or DNA form. DNA includes cDNA, genomic DNA and synthetic DNA; and can be double-stranded or single-stranded, and if it is single-stranded, it can be a coding strand or a non-coding (antisense) strand. The polynucleotide can be in the form of cDNA. The polynucleotide can be a synthetic polynucleotide.

[0406] The present disclosure further relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, a fragment, analog and / or derivative of an antibody or CAR that binds to the tight junction protein 6 of the present disclosure. The present disclosure may provide a polynucleotide comprising 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 a polynucleotide encoding an antibody or CAR that binds to the tight junction protein 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 indicate that the nucleotide sequence of the polynucleotide is identical to the reference sequence except for the following aspects: the polynucleotide sequence may include up to five point mutations in every 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 substituted with another nucleotide, or a plurality of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or between those terminal positions, interspersed between individual nucleotides in the reference sequence, or anywhere in one or more contiguous groups within the reference sequence.

[0407] Polynucleotide variants can contain changes in coding regions, non-coding regions or both. Polynucleotide variants can contain changes that produce silent substitutions, additions or deletions but do not change the properties or activity of the encoded polypeptide. Polynucleotide variants can include silent substitutions that do not result in changes in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, for example, in order to optimize the codon expression of a specific host (i.e., the codons in human mRNA are changed to those preferred by bacterial hosts such as Escherichia coli). Polynucleotide variants can include at least one silent mutation in the non-coding or coding region of the sequence.

[0408] Polynucleotide variants can be produced to regulate or change 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 reduce the expression of the encoded polypeptide. Compared to the parent polynucleotide sequence, the polynucleotide variant can have an increased expression of the encoded polypeptide. Compared to the parent polynucleotide sequence, the polynucleotide variant can have a reduced expression of the encoded polypeptide.

[0409] Also provided are vectors comprising nucleic acid molecules described herein. In one embodiment, nucleic acid molecules can be incorporated into recombinant expression vectors. The present disclosure provides recombinant expression vectors comprising any nucleic acid disclosed herein. As used herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct, when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is in contact with a cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell, allowing mRNA, protein, polypeptide or peptide to be expressed by a host cell. The vectors described herein are not naturally occurring as a whole; however, some vectors may be naturally occurring. The described recombinant expression vectors may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring or non-natural internucleotide bonds, or two types of bonds. Non-naturally occurring or altered nucleotides or internucleotide bonds do not hinder the transcription or replication of the vector.

[0410] The recombinant expression vector of the present disclosure can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host.Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses.Carriers can be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.).Phage vectors such as λGT10, λGT11, λEMBL4, λNM1149 and λZapII (Stratagene) can be used.The example of plant expression vector includes 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 can be a viral vector, for example, a retroviral vector, for example, a gammaretroviral vector.

[0411] Recombinant expression vectors can be prepared using standard recombinant DNA techniques as described in, for example, Sambrook et al., supra and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, SV40, 2μ plasmid, lambda, bovine papilloma virus, and the like.

[0412] Recombinant expression vectors may contain regulatory sequences, such as transcriptional and translational initiation and termination codons, which are specific to the type of host (e.g., bacteria, plants, fungi, or animals) into which the vector is to be introduced, and whether the vector is DNA- or RNA-based, as appropriate.

[0413] The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected hosts. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the expression vector include, for example, neomycin / G418 resistance genes, histidinol x resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0414] The recombinant expression vector may comprise a natural or canonical promoter operably connected to the nucleotide sequence of the present disclosure. The selection of promoters, such as strong promoters, weak promoters, tissue-specific promoters, inducible promoters and development-specific promoters, is within the ordinary skill range of those skilled in the art. Similarly, the combination of nucleotide sequence and promoter is also within the technical scope of those skilled in the art. Promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a RSV promoter, a SV40 promoter or a promoter found in the long terminal repeat of a mouse stem cell virus.

[0415] The recombinant expression vector can be designed for transient expression, stable expression or both. In addition, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0416] In addition, the recombinant expression vector can be made to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that imparts sensitivity to an agent such as a drug to the cell expressing the gene, and causes cell death when the cell contacts or is exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0417] In certain embodiments, the polynucleotide is isolated. The polynucleotide can be substantially pure.

[0418] Host cells containing nucleic acid molecules described herein are also provided. The host cell can be any cell containing heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, the host cell can be a cell from any organism, selected, modified, transformed, grown, used or manipulated in any way, for producing substances by the cell, such as expressing genes, DNA or RNA sequences, proteins or enzymes by the cell. An appropriate host can be determined. For example, a host cell can be selected based on the vector backbone and the desired results. For example, a plasmid or cosmid can be introduced into a prokaryotic host cell to replicate several types of vectors. Bacterial cells such as, but not limited to, DH5α, JM109 and KCB, Competent cells and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. In addition, bacterial cells (such as Escherichia coli LE392) can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include but are not limited to yeast (e.g., YPH499, YPH500 and YPH501), insects and mammals. Examples of mammalian eukaryotic host cells for replication and / or expression of vectors include but are not limited to HeLa, NIH3T3, Jurkat, 293, COS, Saos, PC12, SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Cell Culture Collection (ECACC), Salisbury, Wiltshire, UK, ECACC No.85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.

[0419] 5.6. Pharmaceutical Compositions

[0420] On the one hand, the present disclosure further provides a pharmaceutical composition comprising an antibody (e.g., VHH), a binding molecule comprising an antibody or a therapeutic molecule or an engineered immune effector cell of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an antibody (e.g., VHH provided herein), a binding molecule comprising an antibody or a therapeutic molecule or an engineered immune effector cell of the present disclosure and a pharmaceutically acceptable excipient.

[0421] In some embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of an antibody provided herein (e.g., a VHH provided herein) and a pharmaceutically acceptable excipient. The antibody may be an anti-CLDN6sdAb (e.g., VHH) provided herein, or the antibody may be a combination of an anti-CLDN6sdAb (e.g., VHH) and another antibody (such as GPC3scFv). The antibody may be an agonistic antibody or an antagonistic antibody.

[0422] In some embodiments, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of a therapeutic molecule (such as fusion proteins, immunoconjugates, and multispecific binding molecules) comprising an antibody provided herein and a pharmaceutically acceptable excipient.

[0423] In other embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a CAR comprising an antibody provided herein and a pharmaceutically acceptable excipient. CAR may be a CLDN6CAR provided herein. CAR may be a CLDN6×GPC3 bispecific CAR provided herein, or CAR may be a combination of a CLDN6CAR provided herein and another CAR targeting another antigen (such as GPC3).

[0424] In other embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of an engineered immune effector cell provided herein and a pharmaceutically acceptable excipient. The engineered immune effector cell may include a CLDN6 CAR provided herein. The engineered immune effector cell may include a CLDN6 × GPC3 bispecific CAR provided herein. Alternatively, the engineered immune effector cell may include a combination of a CLDN6 CAR provided herein and another CAR targeting another antigen (such as GPC3).

[0425] In other embodiments, provided herein is a pharm...

Claims

1. An anti-claudin 6 single domain antibody (sdAb), comprising: (i) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO: 7; (ii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO: 8; (iii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:9; (iv) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO: 10; or (v) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO: 11, respectively. 2 . The anti-claudin 6 sdAb of 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. The anti-claudin 6 sdAb according to claim 1 or claim 2, comprising: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a 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. 4 . The anti-Claudin 6 sdAb according to any one of claims 1 to 3 , further comprising one or more FR regions as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO:

11. 5 . The anti-claudin 6 sdAb according to any one of claims 1 to 4 , 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.

6. The anti-claudin 6 sdAb of any one of claims 1 to 4, wherein 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 to 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. The anti-claudin 6 sdAb of claim 1 , wherein the anti-claudin 6 sdAb is a sdAb of camelids. The anti-Claudin 6 sdAb of claim 1 , wherein the anti-Claudin 6 sdAb is a humanized sdAb. 9 . The anti-Claudin 6 sdAb according to claim 1 , wherein the anti-Claudin 6 sdAb is genetically fused or chemically conjugated to a pharmaceutical agent. 10 . The anti-Claudin 6 sdAb according to claim 1 , wherein the anti-Claudin 6 sdAb is fused to an Fc region. The anti-Claudin 6 sdAb of claim 10 , wherein the Fc region is human IgG1 Fc, and wherein optionally the human IgG1 Fc comprises the amino acid sequence of SEQ ID NO:

39. 12 . A fusion protein comprising the anti-claudin 6 sdAb according to claim 1 and human IgG1 Fc, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 21-25.

13. A chimeric antigen receptor (CAR), comprising: (a) an extracellular antigen-binding domain, the extracellular antigen-binding domain comprising the anti-claudin 6 sdAb according to any one of claims 1 to 9; (b) a transmembrane domain; and (c) Intracellular signaling domain.

14. The CAR of claim 13, wherein the extracellular antigen binding domain further comprises one or more additional antigen binding domains, optionally at least one of the additional binding domains binds to GPC3.

15. The CAR of claim 14, wherein the additional antigen binding domain comprises: HCDR1, HCDR2 and HCDR3 having the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively, as shown in VH comprising the amino acid sequence of SEQ ID NO: 18; and LCDR1, LCDR2 and LCDR3 having the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, as shown in VL comprising the amino acid sequence of SEQ ID NO:

19.

16. The CAR of claim 15, wherein 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, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 15; and LCDR3 comprising the amino acid sequence of SEQ ID NO:

17.

17. The CAR of claim 16, wherein the additional antigen binding domain comprises a VH domain and a VL domain, the VH domain comprising 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: 18, and the VL domain comprising 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:

19.

18. The CAR of claim 17, wherein 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.

19. The CAR of claim 18, wherein the additional antigen binding domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

20.

20. The CAR of claim 19, wherein the additional antigen binding domain comprises the amino acid sequence of SEQ ID NO:

20.

21. The CAR of any one of claims 14 to 20, wherein the antigen binding domains are fused to each other via a peptide linker.

22. The CAR of claim 21, wherein the peptide linker is no more than about 50 amino acids long.

23. A CAR as described in any one of claims 13 to 22, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

24. The CAR of claim 23, wherein the transmembrane domain is derived from CD8α.

25. The CAR of any one of claims 13 to 24, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

26. The CAR of claim 25, wherein the primary intracellular signaling domain is derived from CD3ζ.

27. The CAR of claim 25 or claim 26, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain.

28. A CAR as described in claim 27, wherein the co-stimulatory signaling domain is derived from a co-stimulatory molecule, and the co-stimulatory molecule is selected from the group consisting of: ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 and combinations thereof.

29. A CAR as described in claim 28, wherein the co-stimulatory signaling domain is derived from CD137.

30. The CAR of any one of claims 13 to 29, further comprising a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

31. The CAR of claim 30, wherein the hinge domain is derived from CD8α.

32. The CAR of any one of claims 13 to 31, further comprising a signal peptide located at the N-terminus of the polypeptide.

33. The CAR of claim 32, wherein the signal peptide is derived from CD8α.

34. 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. 35 . A nucleic acid comprising a nucleic acid sequence encoding the anti-claudin 6 sdAb of any one of claims 1 to 11 or the fusion protein of claim 12 .

36. A nucleic acid comprising a nucleic acid sequence encoding the CAR of any one of claims 13 to 34.

37. The nucleic acid of claim 36, wherein the nucleic acid further comprises a nucleic acid sequence encoding a chimeric receptor, wherein the chimeric receptor comprises TGFβR and IL23R, optionally wherein the nucleic acid encoding the chimeric receptor comprises the nucleic acid sequence of SEQ ID NO:

33.

38. A chimeric receptor comprising the amino acid sequence of any one of SEQ ID NOs: 32 and 44 to 45.

39. A vector comprising the nucleic acid of any one of claims 35 to 37.

40. An engineered immune effector cell comprising a CAR as described in any one of claims 13 to 34, a chimeric receptor as described in claim 38, a nucleic acid as described in any one of claims 35 to 37, or a vector as described in claim 39.

41. The engineered immune effector cell of claim 40, wherein the immune effector cell is a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, or a combination thereof.

42. A method for producing engineered immune effector cells, comprising introducing the vector of claim 39 into cells.

43. A pharmaceutical composition comprising the anti-claudin 6 sdAb of any one of claims 1 to 11, the nucleic acid of any one of claims 35 to 37, the vector of claim 39, the engineered immune effector cell of any one of claims 40 to 41, and a pharmaceutically acceptable excipient.

44. A method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of the anti-claudin 6 sdAb of any one of claims 1 to 11, the engineered immune effector cell of any one of claims 40 to 41, or the pharmaceutical composition of claim 43.

45. A method of treating a disease or condition in a subject comprising administering to the subject an effective amount of a combination of a CLDN6 antagonist and a GPC3 antagonist, or an antagonist of both CLDN6 and GPC3.

46. ​​The method of claim 44 or claim 45, wherein the disease or disorder is a Claudin 6-associated disease or disorder, and / or a GPC3-associated disease or disorder.

47. The method of claim 46, wherein the disease or condition is cancer.

48. The method of claim 47, wherein the disease or condition is a solid tumor cancer.

49. The method of any one of claims 46 to 48, wherein the disease or condition is selected from the group consisting of germ cell tumors (GCT), ovarian cancer (OC), non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), endometrioid carcinoma (EC), and AFP+ gastric cancer (AFP+GC).

50. The method of any one of claims 44 to 49, wherein the subject is resistant to at least one CLDN6 agent and / or wherein the subject is resistant to at least one GPC3 agent.

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