Constructs targeting labyrinthin or portion thereof and uses thereof

Abstract

To provide anti-labyrinthin constructs as labyrinthin is a useful marker for defining labyrinthin-positive cancers and also a target for treatment based on immunotherapy.SOLUTION: Constructs comprising an antibody moiety that specifically binds to labyrinthin or a portion thereof and an effector domain are provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 789,871, filed January 8, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission, an ASCII text file, are incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 185722000240SEQLIST.TXT, Recorded on: January 7, 2020, Size: 9KB).

[0003] Technical Field The present application relates to constructs comprising an antibody moiety that specifically binds to Labyrinthin or a portion thereof and an effector domain. Methods of making and using these constructs and compositions thereof are also provided. [Background technology]

[0004] background Historically, cancers have been characterized largely based on the tissue type or organ from which they originate, e.g., lung, breast, and colon cancers. Many cancer treatments are also based on tissue- or organ-based classification of cancers. It is well recognized that such tissue- or organ-based classification of cancers may not provide sufficient guidance for the selection of effective treatments. This is due, in part, to the finding that cancers originating from a single tissue type or organ may be highly heterogeneous, and such differences may require individualized cancer treatment approaches. For example, defining cancers by biomarkers, as opposed to tissue type or organ of origin, may allow for improved cancer treatment, e.g., triple-negative breast cancers that lack expression of estrogen receptors, progesterone receptors, and HER2 / neu are not responsive to traditional hormone-based therapies targeting any one or more of the identified receptors and require alternative therapies. After identifying cancer subtypes based on biomarkers, significant research is needed to develop new drugs for the effective treatment of such cancer subtypes.

[0005] One such identified cancer subtype is a cancer that expresses labyrinthin. Labyrinthin is a cell surface protein expressed on the extracellular surface of the cell membrane of some cancers, such as adenocarcinoma. The cell surface expression of labyrinthin is not cell cycle specific. Furthermore, labyrinthin is not found in the serum of normal or tumor-bearing patients, and is not excreted into the culture medium by labyrinthin-positive cell lines.

[0006] Immunotherapy in humans has been limited in part due to adverse reactions to non-human monoclonal antibodies. Early clinical trials using rodent antibodies revealed human anti-mouse antibody (HAMA) and human anti-rat antibody (HARA) responses, which lead to rapid clearance of the antibody. Less immunogenic antibodies have since been developed, including chimeric antibodies, humanized antibodies, PRIMATIZED® antibodies, and human antibodies prepared using transgenic mice or phage display libraries. See, for example, Morrison et al., Proc Natl Acad Sci USA, 81, 1984; and Queen et al., Proc Natl Acad Sci USA, 86, 1989. Avoidance of the HAMA response allows for high doses and repeated administration to achieve a therapeutic response.

[0007] Recent advances in generating mAbs using phage display have made it possible to select agents with exquisite specificity for defined epitopes from large antibody repertoires. Many such mAbs specific for solid tumor antigens have been successfully selected from phage display libraries in the context of HLA-A01 and HLA-A02 (Noy et al., Expert Rev Anticancer Ther, 5, 2005; Chames et al., Proc Natl Acad Sci USA, 97, 2000; Held et al., Eur J Immunol, 34, 2004; Lev et al., Cancer Res, 62, 2002; Klechevsky et al., Cancer Res, 68, 2008). More recently, human mAbs specific for the human WT1 / HLA-A02 complex, a well-described T cell epitope, have been shown to inhibit multiple cancer cell lines and primary cancer cells through Fc-mediated effector cell functions in cellular assays and in vivo models (Dao et al., Sci Transl Med, 5, 2013; Veomett et al., Clin Cancer Res, 2014).

[0008] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the Invention

[0009] overview In one aspect, the present application provides an isolated anti-Labyrinthin construct comprising an antibody portion that specifically binds to Labyrinthin or a portion thereof and an effector domain.

[0010] In some embodiments, the antibody moiety is conjugated to an effector domain. In some embodiments, the effector domain comprises an effector molecule. In some embodiments, the effector molecule comprises a therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid. In some embodiments, the isolated anti-Labyrinthin construct is an antibody drug conjugate (ADC).

[0011] In some embodiments, the effector domain comprises a diagnostic agent. In some embodiments, the diagnostic agent is a label.

[0012] In some embodiments, the antibody moiety is fused to an effector domain.

[0013] In some embodiments, the isolated anti-Labyrinthin construct is a chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antibody portion fused to an effector domain, wherein the effector domain comprises a transmembrane domain and an intracellular signaling domain.

[0014] In some embodiments, the isolated anti-Labyrinthin construct is a bispecific T cell engager (BiTE) that comprises an extracellular domain that comprises an antibody portion fused to an anti-CD3 antibody or fragment thereof.

[0015] In some embodiments, the isolated anti-Labyrinthin construct is a T cell receptor (TCR) comprising an extracellular domain comprising an antibody portion fused to an effector domain, where the effector domain comprises a transmembrane domain and / or an intracellular signaling domain of a TCR subunit.

[0016] In some embodiments, the antibody portion is a full-length antibody, a Fab, a Fab', a (Fab')2, an Fv, or a single chain Fv (scFv). In some embodiments, the antibody portion binds to Labyrinthin or a portion thereof with a Kd of about 0.1 pM to about 500 nM.

[0017] In some embodiments, the antibody portion specifically binds to a labyrinthin-derived peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-32. In some embodiments, the labyrinthin-derived peptide comprises a B cell and a T cell epitope. In some embodiments, the labyrinthin-derived peptide comprises a B cell epitope. In some embodiments, the labyrinthin-derived peptide comprises a T cell epitope.

[0018] In some embodiments, the antibody moiety is multispecific. In some embodiments, the multispecific antibody moiety is a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock-and-lock (DNL) antibody, a chemically cross-linked antibody, a heteromultimeric antibody, or a heteroconjugate antibody. In some embodiments, the multispecific antibody moiety is a tandem scFv comprising two scFvs linked by an optional peptide linker.

[0019] In another aspect, the application provides a pharmaceutical composition comprising any of the isolated anti-Labyrinthin constructs described herein.

[0020] In another aspect, the application provides a host cell expressing any of the isolated anti-Labyrinthin constructs described herein.

[0021] In another aspect, the application provides a nucleic acid encoding a polypeptide component of any of the isolated anti-Labyrinthin constructs described herein.

[0022] In another aspect, the application provides an effector cell comprising any of the nucleic acids described herein. In some embodiments, the effector cell is a T cell.

[0023] In another aspect, the present application provides a method for detecting a cell displaying Labyrinthin or a portion thereof on its surface, the method comprising: (a) contacting the cell with an isolated anti-Labyrinthin construct, wherein the isolated anti-Labyrinthin construct comprises an effector domain that comprises a label; and (b) detecting the presence of the label on the cell.

[0024] In another aspect, the present application provides a method of treating an individual having a Labyrinthin-positive disease, comprising administering to the individual (a) an effective amount of any of the pharmaceutical compositions described herein; or (b) an effective amount of any of the effector cells described herein. In some embodiments, the Labyrinthin status is used as a criterion for selecting individuals for treatment.

[0025] In another aspect, the application provides a method of diagnosing an individual having a Labyrinthin-positive disease, the method comprising: (a) administering to the individual an effective amount of an isolated anti-Labyrinthin construct, wherein the isolated anti-Labyrinthin construct comprises an effector domain that comprises a label; and (b) determining a level of the label in the individual, wherein a level of the label above a threshold level indicates that the individual has a Labyrinthin-positive disease.

[0026] In another aspect, the application provides a method of diagnosing an individual having a Labyrinthin-positive disease, comprising: (a) contacting a sample from the individual with any of the isolated anti-Labyrinthin constructs described herein; and (b) identifying one or more cells in the sample that bind to the isolated anti-Labyrinthin construct, thereby diagnosing the individual having a Labyrinthin-positive disease.

[0027] In some embodiments, the labyrinthin-positive disease is a labyrinthin-positive cancer. In some embodiments, the labyrinthin-positive cancer is an adenocarcinoma. [Brief description of the drawings]

[0028] [Figure 1A-1B] 1A-1B show binding response measurements versus time for the MCA 44-3A6 mouse monoclonal IgG antibody (FIG. 1A) and the X373 scFV derivative (FIG. 1B). [Diagram 2] FIG. 2 shows Western immunoblots of lysates from A549 lung adenocarcinoma cells (positive control) and WI38 fibroblast cells (negative control) using X509Fab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description The present application provides, in some embodiments, isolated constructs (referred to as "anti-Labyrinthin constructs" or "anti-LAB constructs") that comprise an antibody portion that specifically binds to Labyrinthin or a portion thereof and an effector domain. Methods of making and using these constructs and compositions thereof are also provided.

[0030] This application is based in part on the discovery that certain cancers, such as adenocarcinomas, selectively express labyrinthin on cell surface, and labyrinthin-positive cancers do not shed labyrinthin.Furthermore, normal non-cancerous cancers do not express labyrinthin on cell surface.Therefore, labyrinthin is a useful marker for defining labyrinthin-positive cancers and a target for immunotherapy-based treatment.

[0031] Thus, in some embodiments, the present application provides an isolated anti-Labyrinthin construct comprising an antibody portion that specifically binds to Labyrinthin or a portion thereof and an effector domain.

[0032] In some embodiments, the isolated anti-Labyrinthin construct is a chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antibody portion fused to an effector domain, wherein the effector domain comprises a transmembrane domain and an intracellular signaling domain.

[0033] In some embodiments, the isolated anti-Labyrinthin construct is a bispecific T cell engager (BiTE) that comprises an extracellular domain that comprises an antibody portion fused to an anti-CD3 antibody or fragment thereof.

[0034] In some embodiments, the isolated anti-Labyrinthin construct is a T cell receptor (TCR) comprising an extracellular domain comprising an antibody portion fused to an effector domain, where the effector domain comprises a transmembrane domain and / or an intracellular signaling domain of a TCR subunit.

[0035] In some embodiments, the application provides pharmaceutical compositions comprising any of the isolated anti-Labyrinthin constructs described herein.

[0036] In some embodiments, the application provides a host cell expressing any of the isolated anti-Labyrinthin constructs described herein.

[0037] In some embodiments, the application provides nucleic acids encoding any of the polypeptide components of the isolated anti-Labyrinthin constructs described herein.

[0038] In some embodiments, the application provides an effector cell comprising any of the nucleic acids described herein. In some embodiments, the effector cell is a T cell.

[0039] In some embodiments, the present application provides a method for detecting a cell that displays Labyrinthin or a portion thereof on its surface, the method comprising: (a) contacting the cell with an isolated anti-Labyrinthin construct, where the isolated anti-Labyrinthin construct comprises an effector domain that comprises a label; and (b) detecting the presence of the label on the cell.

[0040] In some embodiments, the application provides a method of treating an individual having a Labyrinthin-positive disease, comprising administering to the individual: (a) an effective amount of any of the pharmaceutical compositions described herein; or (b) an effective amount of any of the effector cells described herein.

[0041] In some embodiments, the present application provides a method of diagnosing an individual having a Labyrinthin-positive disease, comprising: (a) administering to the individual an effective amount of an isolated anti-Labyrinthin construct, wherein the isolated anti-Labyrinthin construct comprises an effector domain that comprises a label; and (b) determining a level of the label in the individual, wherein a level of the label above a threshold level indicates that the individual has a Labyrinthin-positive disease.

[0042] In some embodiments, the present application provides a method of diagnosing an individual having a Labyrinthin-positive disease, comprising: (a) contacting a sample from the individual with any of the isolated anti-Labyrinthin constructs described herein; and (b) identifying one or more cells in the sample that bind to the isolated anti-Labyrinthin construct, thereby diagnosing the individual having a Labyrinthin-positive disease.

[0043] It will also be understood by those skilled in the art that modifications of the embodiments and details described herein may be made without departing from the scope of the present disclosure. In addition, although various advantages, aspects, and objectives have been described with reference to various embodiments, the scope of the present disclosure should not be limited by reference to such advantages, aspects, and objectives.

[0044] definition The term "antibody portion" includes full-length antibodies and antigen-binding fragments thereof. Full-length antibodies are composed of two heavy chains and two light chains. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions of both chains usually contain three highly variable loops called complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of a heavy or light chain are inserted between adjacent stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0045] The term "antigen-binding fragment" as used herein refers to antibody fragments including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not constitute a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen that the parent antibody or parent antibody fragment (e.g., parent scFv) binds. An antigen-binding fragment may contain one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0046] The term "epitope" as used herein refers to the particular grouping of atoms or amino acids on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions may bind to the same epitope in an antigen if they exhibit competitive binding to the antigen.

[0047] As used herein, a first antibody portion "competes" with a second antibody portion for binding to a target labyrinthin if the first antibody portion inhibits target labyrinthin binding of the second antibody portion by at least about 50% (such as at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of an equimolar concentration of the first antibody portion, and vice versa. A high-throughput process for "binning" antibodies based on their cross-competition is described in PCT Publication No. WO03 / 48731.

[0048] As used herein, the term "specifically binds to" or "specific for" refers to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody portion, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody portion that specifically binds to a target (which may be an epitope) is an antibody or antibody portion that binds to this target with higher affinity, avidity, more readily, and / or longer duration than it binds to other targets. In some embodiments, an antibody or antibody portion that specifically binds to an antigen reacts with one or more antigenic determinants of the antigen (e.g., labyrinthin or a portion thereof) with a binding affinity that is at least about 10 times higher than its binding affinity to other targets.

[0049] As used herein, an "isolated" anti-Labyrinthin construct refers to an anti-Labyrinthin construct that is (1) not associated with proteins found in nature, (2) free of other proteins from the same source, (3) expressed by cells from a different species, or (4) not naturally occurring.

[0050] As used herein, the term "isolated nucleic acid" is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin, or some combination thereof, and by virtue of its origin, an "isolated nucleic acid" is: (1) not associated with all or a portion of a polynucleotide with which it is found in nature; (2) operably linked to a polynucleotide with which it is not linked in nature; or (3) not found in nature as part of a larger sequence.

[0051] The term "CDR" or "complementarity determining region" as used herein is intended to mean the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); and Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison.

[0052] TIFF2025060688000001.tif73170

[0053] The term "chimeric antibody" refers to antibodies in which a portion of the heavy and / or light chains are identical to or homologous to sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to sequences in antibodies derived from another species or belonging to another antibody class or subclass, and to fragments of such antibodies so long as they exhibit the biological activity of this invention (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0054] The term "semi-synthetic" with reference to an antibody or antibody portion means that the antibody or antibody portion has one or more naturally occurring sequences and one or more non-naturally occurring (i.e., synthetic) sequences.

[0055] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the heavy and light chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0056] A "single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0057] The term "diabody" refers to small antibody fragments prepared by constructing scFv fragments (see previous paragraph) that typically have a short linker (such as about 5 to about 10 residues) between the VH and VL domains, thereby achieving interchain rather than intrachain pairing of the V domains, thereby creating a bivalent fragment, i.e., a fragment with two antigen binding sites. Bispecific diabodies are heterodimers of two "crossover" Fv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in further detail, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0058] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can include residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0059] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR of the present invention is one that binds an IgG antibody (gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review M in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). The term includes the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and / or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal 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)).

[0060] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain noncovalently bound to β2-microglobulin. The multiple functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays a role in the passive delivery of immunoglobulin IgG from mother to child and in the regulation of serum IgG levels. FcRn acts as a salvage receptor, binding and transporting pinocytosed IgG in an intact form both intracellularly and intercellularly, rescuing them from the default degradative pathway.

[0061] The "CH1 domain" (also called "C1" for "H1" domain) of a human IgG Fc region typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0062] The "hinge region" is generally defined as the stretch from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain S-S bonds in the same positions.

[0063] The "CH2 domain" (also called "C2" for "H2" domain) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact native IgG molecules. It has been speculated that the carbohydrates may provide a substitute for domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

[0064] The "CH3 domain" (also called the "C2" or "H3" domain) comprises the stretch of the Fc region from the C-terminal residue to the CH2 domain (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically to amino acid residue 446 or 447 for IgG).

[0065] A "functional Fc fragment" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions generally require the Fc region to bind to a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays known in the art.

[0066] An antibody having a variant IgG Fc with "altered" FcR binding affinity or ADCC activity is an antibody that has enhanced or decreased FcR binding activity (e.g., FcγR or FcRn) and / or ADCC activity compared to a parent polypeptide or a polypeptide comprising a native-sequence Fc region. A variant Fc that "exhibits increased binding" to an FcR has a higher affinity (e.g., a lower apparent K) than the parent polypeptide or native-sequence IgG Fc. d or IC 50 In some embodiments, the improvement in binding compared to the parent polypeptide is about 3-fold, e.g., about 5, 10, 25, 50, 60, 100, 150, 200, or up to 500-fold, or about a 25%-1000% improvement in binding. A polypeptide variant that "exhibits reduced binding" to an FcR has a lower affinity (e.g., a higher apparent K value) than the parent polypeptide. d or higher IC 50 The reduction in binding compared to the parent polypeptide can be about a 40% or greater reduction in binding.

[0067] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxic agents. Antibodies "arm" the cytotoxic cells and are absolutely necessary for such killing. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0068] A polypeptide comprising a variant Fc region that "exhibits increased ADCC" or mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of human effector cells more effectively than a polypeptide having a wild-type IgG Fc or a parent polypeptide is one that is substantially more effective at mediating ADCC in vitro or in vivo when the amounts of the polypeptide having the variant Fc region and the polypeptide having the wild-type Fc region (or parent polypeptide) in the assay are essentially the same. Generally, such variants will be identified using any in vitro ADCC assay known in the art, such as an assay or method for measuring ADCC activity, e.g., in an animal model. In some embodiments, the variant is about 5-fold to about 100-fold, e.g., about 25-fold to about 50-fold more effective at mediating ADCC than the wild-type Fc (or parent polypeptide).

[0069] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that is bound to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Pat. No. 6,194,551 (B1) and WO 99 / 51642. The contents of those patent publications are specifically incorporated herein by reference. See also Idusogie et al. J. Immunol.164: 4178-4184 (2000).

[0070] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. A phrase nucleotide sequence that encodes a protein or RNA may also contain introns to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0071] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein coding regions, are in the same reading frame.

[0072] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of two compared sequences are occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is performed when the two sequences are aligned to give maximum homology.

[0073] The term "substantially homologous" as used herein refers to the sequence similarity of the sequence disclosed herein when compared with a reference sequence, where the sequence has at least about 85% similarity (e.g., homology) with the reference or a portion thereof, for example, at least about 86% similarity, 87% similarity, 88% similarity, 89% similarity, 90% similarity, 91% similarity, 92% similarity, 93% similarity, 94% similarity, 95% similarity, 96% similarity, 97% similarity, 98% similarity, 99% similarity, or 100% similarity. Methods for determining sequence similarity are known in the art and are described, for example, in Pearson, WR, Curr Protoc Bioinformatics, 2013.

[0074] The term "label" as used herein refers to a detectable compound or composition that can be directly or indirectly conjugated to an anti-Labyrinthin antibody moiety. The label can be detectable in itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze chemical alteration of a substrate compound or composition that is detectable.

[0075] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms attributable to the disease, alleviation of the extent of the disease, stabilization of the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the disease state, providing remission of the disease (partial or total disease), reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, increasing or improving the quality of life, and / or prolonging survival. "Treatment" also includes the reduction of pathological consequences of cancer (e.g., tumor volume, etc.). The methods of the invention contemplate any one or more of these aspects of treatment.

[0076] The terms "recurrence," "relapse," or "relapsed" refer to the recurrence of cancer or disease after clinical assessment of disappearance of the disease. A diagnosis of distant metastasis or local recurrence may be considered a recurrence.

[0077] The terms "refractory" or "resistant" refer to a cancer or disease that has not responded to treatment.

[0078] "Activated" as used herein in reference to T cells refers to a state of T cells that has been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function.

[0079] The term "effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as ameliorating, alleviating, reducing, and / or delaying one or more symptoms of the disorder, condition, or disease. With respect to cancer, an effective amount includes, for example, an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (such as inhibiting tumor growth), or prevent or delay other undesirable cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay the onset of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. In the case of cancer, an effective amount of an agent or composition can (i) reduce the number of cancerous cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent, and preferably stop, cancer cell invasion into peripheral organs; (iv) inhibit (e.g., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0080] As used herein, "pharmacologically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. A pharma-ceutically acceptable carrier or excipient preferably meets the required standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0081] As used herein, "delaying" the onset of cancer means to postpone, prevent, slow, retard, stabilize, and / or prolong the onset of the disease. Such delays can be of various lengths of time, depending on the disease being treated and / or the individual's medical history. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that "delays" the onset of cancer is one that reduces the likelihood of disease onset in a given time frame and / or reduces the extent of disease in a given time frame, compared to not using the method. Such comparisons are typically based on clinical trials using a statistically significant number of subjects. The onset of cancer can be detected using standard methods, including, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset may also be undetectable at first, and may refer to the progression of cancer, including onset, recurrence, and onset.

[0082] The term "individual" refers to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate.

[0083] The term "based on" or "criterion of" as used herein includes evaluating, determining, obtaining, or measuring one or more characteristics of an individual or a cancer therein as described herein, and in some embodiments, selecting an individual suitable for receiving the treatment described in the methods disclosed herein. For example, when the labyrinthine status of a cancer is used as a criterion for selecting an individual for the treatment method described herein, evaluating (or assisting in the evaluation), measuring, obtaining, or determining the labyrinthine status can be included in the treatment method described herein, for example, the labyrinthine status is measured before and / or during and / or after treatment, and the obtained value is used by the clinician in evaluating any of the following: (a) high likelihood or likelihood that an individual is suitable for receiving treatment initially; (b) high likelihood or likelihood that an individual is not suitable for receiving treatment initially; (c) responsiveness to treatment; (d) high likelihood or likelihood that an individual is suitable for continuing to receive treatment; (e) high likelihood or likelihood that an individual is not suitable for continuing to receive treatment; (f) dosage adjustment; or (g) prediction of possible clinical benefit.

[0084] The criteria disclosed herein, e.g., labyrinthin status, used with the methods of the present application, may in some aspects be based on a comparison to a control. In some embodiments, the control is a known standard obtained from the literature (e.g., a known gene sequence, RNA sequence, protein sequence, gene expression level). In some embodiments, the control is a control sample obtained from an individual to be treated or being treated using the methods disclosed herein (e.g., a control sample from a non-cancerous tissue). In some embodiments, the control is a control sample obtained from an individual other than the individual to be treated or being treated using the methods disclosed herein (e.g., a control sample from a healthy volunteer or a volunteer without cancer). In some embodiments, the control is obtained from a given patient population. For example, with respect to the level of gene expression or enzyme activity level, the control level can be the median expression level of that gene for the patient population, or the median enzyme activity level of that enzyme. And, for example, if the expression level of the gene of interest of one patient is determined to be above the median expression level of the patient population, the patient is determined to have high expression of the gene of interest. Alternatively, if the expression level of the gene of interest in a patient is determined to be lower than the median expression level of a patient population, the patient is determined to have low expression of the gene of interest. In some embodiments, the patient has a disease (such as cancer) and the patient population does not have the disease. In some embodiments, the patient and the patient population have the same tissue type of disease. The population can be about 2, 5, 10, 15, 20, 25, 30, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, or at least about 2, 5, 10, 15, 20, 25, 30, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500 in terms of the number of individuals measured. Preferably, a sufficient number of individuals are measured to provide a statistically significant population that can be determined by methods known in the art, hi some embodiments, the population is a group participating in a clinical trial.

[0085] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and their grammatical equivalents, are intended to be equivalent in meaning and open ended in that the item or items following any one of these words are not meant to imply a limited enumeration of such item or items, but are not meant to be limited to only the item or items listed. For example, an article "comprising" components A, B, and C may be composed of (i.e., include only) components A, B, and C, or may include not only components A, B, and C, but also one or more other components. As such, "comprising" and its similar forms and their grammatical equivalents are intended and understood to include disclosure of embodiments that "consist essentially of" or "consist of."

[0086] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included within the disclosure, subject to any specifically excluded limit in the stated range, unless the context clearly dictates otherwise. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0087] Reference herein to a value or parameter preceded by "about" includes (and describes) the variation relative to the value or parameter itself. For example, a reference to "about X" includes the reference to "X."

[0088] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0089] Anti-LAB construct The present application provides anti-labyrinthin constructs, such as isolated anti-LAB constructs (herein referred to as "anti-LAB constructs"), comprising an antibody moiety that specifically binds to labyrinthin or a portion thereof and an effector domain. In some embodiments, the antibody moiety is conjugated to the effector domain. For example, the effector domain may comprise an effector molecule, which may be a therapeutic agent or a label. In some embodiments, the therapeutic agent is selected from the group consisting of a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid. In some embodiments, the effector molecule is a diagnostic agent, such as a label. Conjugation of effector molecules is well known in the art and may include covalent bonds (e.g., via chemical bonds or via linkers) or non-covalent interactions (e.g., via biotin / streptavidin and other protein-protein interaction pairs).

[0090] In some embodiments, the anti-Labyrinthin construct is a fusion protein. For example, the anti-Labyrinthin antibody moiety can be part of a CAR (chimeric antigen receptor), an engineered TCR, or a bispecific T cell engager molecule. These various constructs are discussed in more detail in the following sections.

[0091] Anti-LAB CAR In one aspect, the present application provides a chimeric antigen receptor (CAR) comprising an antibody portion that specifically binds to Labyrinthin or a portion thereof (referred to herein as an "anti-LAB CAR"). Also provided is a CAR effector cell (e.g., a T cell) comprising a CAR comprising an anti-LAB antibody portion (also referred to herein as an "anti-LAB CAR effector cell", e.g., an "anti-LAB CAR T cell").

[0092] The anti-LAB CAR comprises (a) an extracellular domain comprising an anti-LAB antibody portion that specifically binds to Labyrinthin, and (b) an intracellular signaling domain. A transmembrane domain may be present between the extracellular domain and the intracellular domain. A linker or spacer domain may be present between the extracellular domain and the transmembrane domain of the anti-LAB CAR, or between the extracellular domain and the transmembrane domain of the anti-LAB CAR. The linker or spacer spacer domain may be any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular or intracellular domain of a polypeptide chain. The spacer domain may comprise up to about 300 amino acids, including, for example, from about 10 to about 100, or from about 25 to about 50 amino acids.

[0093] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region of particular use in the present invention may be derived from (i.e., comprise at least the transmembrane region of) the α, β, δ or γ chain of the T-cell receptor, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. In some embodiments, the transmembrane domain may be synthetic, in which case it may comprise primarily hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan and valine may be found at each end of the synthetic transmembrane domain. In some embodiments, for example, a short oligo- or polypeptide linker having a length between about 2 and about 10 (e.g., about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in length can form the linkage between the transmembrane domain and the intracellular signaling domain of the anti-LAB CAR. In some embodiments, the linker is a glycine-serine doublet.

[0094] In some embodiments, a transmembrane domain is used that is naturally associated with one of the sequences of the intracellular domain of the anti-LAB CAR (e.g., if the anti-LAB CAR intracellular domain comprises a CD28 costimulatory sequence, the transmembrane domain of the anti-LAB CAR is derived from the CD28 transmembrane domain). In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of the domain to the transmembrane domain of the same or a different surface membrane protein in order to minimize interactions with other members of the receptor complex.

[0095] The intracellular signaling domain of the anti-LAB CAR is involved in the activation of at least one of the normal effector functions of the immune cell in which the anti-LAB CAR is placed. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term "intracellular signaling sequence" is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0096] Examples of intracellular signaling domains for use in the anti-LAB CARs of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capability.

[0097] In some embodiments, it is known that signals generated through the TCR alone are insufficient for the complete activation of T cells, and that secondary or costimulatory signals may also be required. Thus, T cell activation may in some embodiments be mediated by two different classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (costimulatory signaling sequences). Primary signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. The anti-LAB CAR construct in some embodiments contains one or more ITAMs. Examples of ITAMs that contain primary signaling sequences that are particularly used in the present invention include those derived from TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0098] In some embodiments, the anti-LAB CAR comprises a primary signaling sequence derived from CD3zeta. For example, the intracellular signaling domain of the CAR can comprise a CD3zeta intracellular signaling sequence, either by itself or in combination with any other desired intracellular signaling sequence useful in the context of the anti-LAB CAR of the present invention. For example, the intracellular domain of the anti-LAB CAR can comprise a CD3zeta intracellular signaling sequence and a costimulatory signaling sequence. The costimulatory signaling sequence can be part of the intracellular domain of a costimulatory molecule, including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like.

[0099] In some embodiments, the intracellular signaling domain of the anti-LAB CAR comprises the intracellular signaling sequence of CD3zeta and the intracellular signaling sequence of CD28. In some embodiments, the intracellular signaling domain of the anti-LAB CAR comprises the intracellular signaling sequence of CD3zeta and the intracellular signaling sequence of 4-1BB. In some embodiments, the intracellular signaling domain of the anti-LAB CAR comprises the intracellular signaling sequence of CD3zeta and the intracellular signaling sequences of CD28 and 4-1BB.

[0100] Thus, for example, in some embodiments, an anti-LAB CAR is provided that comprises (a) an extracellular domain comprising an anti-LAB antibody portion that specifically binds to Labyrinthin, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the intracellular signaling domain can activate an immune cell. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3 zeta intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3 zeta intracellular signaling sequence and a CD28 intracellular signaling sequence.

[0101] Anti-LAB TCR In some embodiments, the anti-LAB antibody portion is fused to the N-terminus of a T cell receptor (TCR) subunit, thus forming an anti-LAB T cell receptor (herein referred to as an "anti-LAB TCR").

[0102] As used herein, "T cell receptor" or "TCR" refers to an endogenous or recombinant T cell receptor that comprises an extracellular antigen-binding domain that binds to a specific antigenic peptide bound in an MHC molecule. In some embodiments, the TCR comprises a TCRα polypeptide chain and a TCRβ polypeptide chain. In some embodiments, the TCR comprises a TCRγ polypeptide chain and a TCRδ polypeptide chain. In some embodiments, the TCR specifically binds to a tumor antigen. "TCR-T" refers to a T cell that expresses a recombinant TCR. As used herein, "TCR complex" refers to a complex of TCR and CD3. As used herein, "TCR subunit" refers to a subunit of the TCR complex, including, for example, TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, and CD3ζ. The anti-LAB antibody moiety can be fused to the N-terminus of any of the TCR subunits.

[0103] Thus, for example, in some embodiments, an engineered TCR is provided that comprises (a) an anti-LAB antibody portion that specifically binds to Labyrinthin, and (b) a TCR subunit. In some embodiments, the antibody portion is fused to the N-terminus of the TCR subunit. In some embodiments, the TCR subunit is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, and CD3ζ.

[0104] Anti-Labyrinthin Bispecific Antibody In some embodiments, the anti-LAB construct is a multispecific antibody. Antibodies or antigen-binding fragments thereof with different specificities can also be chemically crosslinked to generate multispecific heteroconjugate antibodies. For example, two F(ab')2 molecules, each with specificity for a different antigen, can be chemically linked.

[0105] In some embodiments, the antibody moiety is multispecific. In some embodiments, the multispecific antibody moiety is a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock-and-lock (DNL) antibody, a chemically cross-linked antibody, a heteromultimeric antibody, or a heteroconjugate antibody. In some embodiments, the multispecific antibody moiety is a tandem scFv comprising two scFvs linked by an optional peptide linker.

[0106] In some embodiments, multispecific antibodies can be prepared using recombinant DNA technology. For example, bispecific antibodies can be designed by fusing two scFvs into tandem scFvs, e.g., fusing two scFvs via a peptide linker. One example of a tandem scFv is a bispecific T cell engager. Bispecific T cell engagers are created by linking an anti-CD3 scFv to an scFv specific for a surface antigen of a target cell, such as a tumor-associated antigen (TAA), resulting in the redirection of T cells to the target cell.

[0107] Thus, for example, in some embodiments, a bispecific T cell engager is provided that comprises (a) an anti-LAB antibody portion that specifically binds to Labyrinthin, and (b) an anti-CD3 antibody portion that specifically binds to CD3. In some embodiments, a bispecific T cell engager is provided that comprises (a) an scFv that specifically binds to Labyrinthin, and b) an scFv that specifically binds to CD3.

[0108] Anti-LAB antibody-drug conjugates (ADCs) or other conjugates Anti-LAB constructs, in some embodiments, include immunoconjugates (also referred to herein as "anti-LAB immunoconjugates") that include an anti-LAB antibody moiety attached to an effector molecule. In some embodiments, the effector molecule is a therapeutic agent, such as a cancer therapeutic agent, that is cytotoxic, cytostatic, or otherwise provides some therapeutic benefit. In some embodiments, the effector molecule is a label that can generate, directly or indirectly, a detectable signal.

[0109] In some embodiments, an anti-LAB immunoconjugate is provided that comprises an anti-LAB antibody moiety and a therapeutic agent (also referred to herein as an "antibody drug conjugate" or "ADC"). In some embodiments, the therapeutic agent is a toxin that is cytotoxic, cytostatic, or otherwise prevents or reduces the ability of a target cell to divide. Importantly, since most normal cells do not display LAB on their surface, they cannot bind to the anti-LAB immunoconjugate and are protected from the killing effects of toxins or other therapeutic agents.

[0110] Therapeutic agents used in anti-LAB immunoconjugates include, for example, daunomycin, doxorubicin, methotrexate, and vindesine (see, for example, Rowland et al., Cancer Immunol Immunother, 21, 1986). Toxins used in anti-LAB immunoconjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, and small molecule toxins such as geldanamycin, maytansinoids, and calicheamicin. Toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic agents tend to become inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0111] In some embodiments, the anti-LAB antibody moiety can be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, where the antibody-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0112] The present application further provides anti-LAB immunoconjugates comprising an anti-LAB antibody moiety attached to an effector molecule, where the effector molecule is a label capable of generating a detectable signal, either indirectly or directly. These anti-LAB immunoconjugates can be used in research or diagnostic applications, such as in vivo detection of cancer. The label is preferably capable of generating a detectable signal, either directly or indirectly. For example, the label can be a radiopaque or radioactive isotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, β-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. In some embodiments, the anti-LAB immunoconjugate is indirectly detectable. For example, an anti-LAB immunoconjugate can be detected using a secondary antibody that is specific for the anti-LAB immunoconjugate and contains a detectable label.

[0113] Thus, for example, in some embodiments, an anti-LAB immunoconjugate is provided, comprising: (a) an anti-LAB antibody portion that specifically binds to Labyrinthin; and (b) an effector molecule. In some embodiments, the effector molecule is a therapeutic agent. In some embodiments, the effector molecule is a label.

[0114] Anti-LAB antibody part In one aspect, the present application provides an antibody moiety (herein referred to as an "anti-LAB antibody moiety") that specifically binds to Labyrinthin or a portion thereof. An anti-Labyrinthin construct comprises an anti-LAB antibody moiety. In some embodiments, the anti-LAB antibody moiety specifically binds to Labyrinthin present on the surface of a cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is in a solid tumor, such as a metastatic cancer cell.

[0115] In some embodiments, the anti-LAB antibody portion is a full-length antibody. In some embodiments, the anti-LAB antibody portion is an antigen-binding fragment, such as an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), and single-chain antibody molecule (scFv). In some embodiments, the anti-LAB antibody portion is an scFv. In some embodiments, the anti-LAB antibody portion is human, humanized, or semi-synthetic.

[0116] In some embodiments, the anti-LAB antibody portion specifically binds to a labyrinthin-derived peptide or a portion thereof. In some embodiments, the labyrinthin-derived peptide comprises an amino acid sequence selected from a portion of labyrinthin (SEQ ID NO: 1; see Table 2). In some embodiments, the anti-LAB antibody portion is derived from an antibody produced by using a labyrinthin-derived peptide as an immunogen. In some embodiments, when a labyrinthin-derived peptide is used as an immunogen, the labyrinthin-derived peptide is conjugated to a carrier such as albumin or KLH.

[0117] In some embodiments, a labyrinthin-derived peptide is a peptide having sequence similarity of at least about 60% to a portion of labyrinthin (SEQ ID NO:1), such as at least about 65% similarity, 70% similarity, 75% similarity, 80% similarity, 85% similarity, 90% similarity, or 95% similarity, where the labyrinthin-derived peptide does not include a terminal proline residue, and where the labyrinthin-derived peptide includes at least one proline residue, such as 2, 3, 4, or 5 proline residues. In some embodiments, a labyrinthin-derived peptide is a peptide having sequence similarity of at least about 60% to a portion of labyrinthin (SEQ ID NO:1), such as at least about 65% similarity, 70% similarity, 75% similarity, 80% similarity, 85% similarity, 90% similarity, or 95% similarity, where one end of the labyrinthin-derived peptide does not include a terminal proline residue, and where the labyrinthin-derived peptide includes at least one proline residue, such as 2, 3, 4, or 5 proline residues. In some embodiments, the labyrinthin-derived peptide is 7-50 amino acids in length, e.g., 7-25 amino acids in length, 7-13 amino acids in length, or 21-25 amino acids in length. In some embodiments, the labyrinthin derived peptide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some embodiments, the labyrinthin derived peptide is substantially homologous to a portion of labyrinthin (SEQ ID NO: 1). In some embodiments, the labyrinthin-derived peptide has a sequence similarity to a portion of labyrinthin (SEQ ID NO:1) of at least about 60% similarity, such as at least 65% similarity, 70% similarity, 75% similarity, 80% similarity, 85% similarity, 90% similarity, or 95% similarity.The labyrinthin-derived peptide or derivative thereof has a sequence similarity of at least about 60% similarity to a portion of labyrinthin (SEQ ID NO: 1), where 1, 2, 3, 4 of the 5 amino acids of the sequence of the labyrinthin-derived peptide are deleted, substituted, inserted, and / or added to the labyrinthin-derived peptide, where, when there is a substitution, insertion, and / or addition, a portion is substituted, inserted, and / or added to the sequence. In some embodiments, the substituted, inserted, or added portion is a natural amino acid (e.g., an α-amino acid or an L-amino acid or a D-amino acid) or a non-natural amino acid. In some embodiments, the substituted, inserted, or added portion is an amino acid substitution or a linker. In some embodiments, the labyrinthin-derived peptide comprises a T-cell epitope and / or a B-cell epitope.

[0118] In some embodiments, the labyrinthin-derived peptide comprises a sequence selected from SEQ ID NOs: 2-32 (Table 2), or a variant thereof. In some embodiments, the labyrinthin-derived peptide comprising a sequence selected from SEQ ID NOs: 2-32 (Table 2), or a variant thereof, comprises one or two flanking amino acid sequences, which are added to the ends of the core sequence provided in SEQ ID NOs: 2-32. In some embodiments, a labyrinthin-derived peptide comprising one or two flanking amino acid sequences is a peptide having a sequence similarity of at least about 60% to a portion of labyrinthin (SEQ ID NO: 1), such as at least 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the flanking amino acid sequences are based on the sequence of a portion of labyrinthin (SEQ ID NO: 1), and are each continuations of the labyrinthin sequence from each of the core sequences provided in SEQ ID NOs: 2-32, for example from one or two ends of the core sequence. For example, for SEQ ID NO: 3, the first flanking amino acid sequence of the two amino acids to the left of SEQ ID NO: 3 would be Pro-Ala, and the second flanking amino acid sequence of the two amino acids to the right of SEQ ID NO: 3 would be Glu-Ala.

[0119] TIFF2025060688000002.tif227170TIFF2025060688000003.tif74170

[0120] In some embodiments, the anti-LAB antibody moiety (or the anti-LAB construct comprising the anti-LAB antibody moiety) has a K d The antibody binds to labyrinthin or a portion thereof at about 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any range between these values.

[0121] In some embodiments the anti-LAB antibody moiety (or an anti-LAB construct comprising an anti-LAB antibody moiety) binds to one or more epitopes of Labyrinthin, such as two or three epitopes or parts thereof.

[0122] In some embodiments, the anti-LAB antibody moiety comprises a specific sequence or a specific variant of such a sequence. In some embodiments, the amino acid substitution in the variant sequence does not substantially reduce the ability of the anti-LAB antibody moiety to bind to Labyrinthin or a part thereof. For example, modifications may be made that do not substantially reduce Labyrinthin binding affinity. Modifications that substantially improve Labyrinthin binding affinity or affect some other properties, such as specificity and / or cross-reactivity with related variants of Labyrinthin, are also contemplated.

[0123] Anti-labyrinthin antibodies have been disclosed in the art, e.g., U.S. Pat. No. 6,166,176, U.S. Pat. No. 7,635,759, and WO2011116014, the disclosures of each of which are incorporated herein by reference in their entirety. In some embodiments, the anti-LAB antibody moiety comprises MCA44-3A6 or a portion thereof that binds to labyrinthin. In some embodiments, the anti-LAB antibody moiety is MCA44-3A6. In some embodiments, the anti-LAB antibody moiety competes with MCA44-3A6 or a portion thereof that binds to labyrinthin. In some embodiments, the anti-LAB antibody moiety comprises one or more CDRs from MCA44-3A6. In some embodiments, the anti-LAB antibody moiety comprises X373 or a portion thereof that binds to labyrinthin. In some embodiments, the anti-LAB antibody moiety is X373. In some embodiments, the anti-LAB antibody moiety competes with X373 or a portion thereof that binds to labyrinthin. In some embodiments, the anti-LAB antibody portion comprises X509 or a portion thereof that binds to labyrinthin. In some embodiments, the anti-LAB antibody portion is X509. In some embodiments, the anti-LAB antibody portion competes with X509 or a portion thereof that binds to labyrinthin. In some embodiments, the anti-LAB antibody portion comprises one or more of the variable heavy domain complementarity determining regions (CDRs) set forth in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. In some embodiments, the anti-LAB antibody portion comprises one or more of the variable light domain complementarity determining regions (CDRs) set forth in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38. In some embodiments, the anti-LAB antibody portion comprises one or more of the variable heavy domain complementarity determining regions (CDRs) set forth in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35 and one or more of the variable light domain complementarity determining regions (CDRs) set forth in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38.

[0124] TIFF2025060688000004.tif54170

[0125] Nucleic Acids and Effector Cells Nucleic acid molecules encoding anti-LAB constructs or anti-LAB antibody portions are also provided herein. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a full-length anti-LAB antibody is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a multispecific anti-LAB molecule (e.g., a multispecific anti-LAB antibody, a bispecific anti-LAB antibody, or a bispecific T cell engager anti-LAB antibody), or a polypeptide portion thereof, is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding an anti-LAB antibody portion is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding an anti-LAB immunoconjugate, or a polypeptide portion thereof, is provided.

[0126] The present application also includes variants to these nucleic acid sequences, for example, variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the anti-LAB constructs or anti-LAB antibody portions of the present application under at least moderately stringent hybridization conditions.

[0127] The present invention also provides a vector into which the nucleic acid of the present invention is inserted.

[0128] Briefly, expression of an anti-LAB construct (e.g., an anti-LAB CAR) or a polypeptide portion thereof by a natural or synthetic nucleic acid encoding the anti-LAB construct or a polypeptide portion thereof can be achieved by inserting the nucleic acid into an appropriate expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including, for example, a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR). The vector can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0129] The nucleic acids of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466. These are incorporated by reference in their entireties. In some embodiments, the present invention provides gene therapy vectors.

[0130] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0131] Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0132] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of interest either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many retroviral systems are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of the transgene and propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of being less immunogenic.

[0133] Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but recently, many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

[0134] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of the polynucleotide sequence to which it is operably linked when expression of the polynucleotide sequence is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0135] The expression vector introduced into the cell to evaluate the expression of the polypeptide or a part thereof can also contain either a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via the viral vector. In other embodiments, the selectable marker can be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include antibiotic resistance genes, such as neo.

[0136] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, reporter genes are genes that encode a polypeptide that is not present or expressed in the recipient organism or tissue and whose expression is indicated by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tel et al., FEBS Letters, 479, 2000). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such a promoter region can be linked to the reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0137] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vectors can be transferred into the host cells by physical, chemical, or biological means.

[0138] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, the introduction of a polynucleotide into a host cell is by calcium phosphate transfection.

[0139] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus 1, adenoviruses and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0140] Chemical means for introducing polynucleotides into host cells include polymer complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0141] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations for the introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo) is contemplated. In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained in suspension in lipid, contained or complexed with micelles, or otherwise associated with lipid. The lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be interspersed in the solution, forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons, such as fatty acids, alcohols, amines, aminoalcohols, aldehydes, and their derivatives.

[0142] Regardless of the method used to introduce exogenous nucleic acid into a host cell or otherwise expose the cell to an inhibitor of the invention, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blot) or by the assays described herein to identify substances that fall within the scope of the present invention.

[0143] In some embodiments, an effector cell (such as a T cell) is provided that displays an anti-LAB construct as described herein on its surface. In some embodiments, the effector cell (such as a T cell) comprises an anti-LAB CAR polypeptide as described herein on its surface. In some embodiments, the effector cell comprises a nucleic acid encoding an anti-LAB CAR polypeptide as described herein, wherein the anti-LAB CAR polypeptide expressed from the nucleic acid, or a portion thereof, is localized on the surface of the effector cell. In some embodiments, the effector cell (such as a T cell) comprises an anti-LAB TCR polypeptide as described herein on its surface. In some embodiments, the effector cell comprises a nucleic acid encoding an anti-LAB TCR polypeptide as described herein, wherein the anti-LAB TCR polypeptide expressed from the nucleic acid, or a portion thereof, is localized on the surface of the effector cell. In some embodiments, the effector cell (such as a T cell) comprises a multispecific antibody, such as a bispecific antibody, a polypeptide as described herein on its surface. In some embodiments, the effector cell comprises a nucleic acid encoding a multispecific antibody, such as a bispecific antibody, polypeptide described herein, wherein the multispecific antibody, such as a bispecific antibody, polypeptide, or portion thereof expressed from the nucleic acid is localized on the surface of the effector cell.

[0144] Exemplary effector cells useful in the present invention include, but are not limited to, dendritic cells (including immature and mature dendritic cells), T lymphocytes (naive T cells, effector T cells, memory T cells, cytotoxic T lymphocytes, helper T cells, natural killer T cells, Treg cells, tumor infiltrating lymphocytes (TILs), and lyphokine-activated killer (LAK) cells), B cells, natural killer (NK) cells, monocytes, macrophages, neutrophils, granulocytes, and combinations thereof. Subpopulations of effector cells can be defined by the presence or absence of one or more cell surface markers known in the art (e.g., CD3, CD4, CD8, CD19, CD20, CD11c, CD123, CD56, CD34, CD14, CD33, etc.).

[0145] In some embodiments, the effector cells are engineered effector cells, such as engineered mammalian effector cells. When the pharmaceutical composition comprises a plurality of engineered mammalian effector cells, the engineered mammalian effector cells can be a specific subpopulation of an effector cell type, a combination of subpopulations of an effector cell type, or a combination of two or more effector cell types. In some embodiments, the effector cells are present in a homogenous cell population. In some embodiments, the effector cells are present in a heterogeneous cell population that is enriched in effector cells. In some embodiments, the engineered mammalian cells are lymphocytes. In some embodiments, the engineered mammalian cells are not lymphocytes. In some embodiments, the engineered mammalian cells are suitable for adoptive immunotherapy. In some embodiments, the engineered mammalian cells are PBMCs. In some embodiments, the engineered mammalian cells are effector cells derived from PBMCs. In some embodiments, the engineered mammalian cells are T cells. In some embodiments, the engineered mammalian cells are CD4+ T cells. In some embodiments, the engineered mammalian cells are CD8+ T cells. In some embodiments, the engineered mammalian cell is a B cell. In some embodiments, the engineered mammalian cell is a NK cell.

[0146] In some embodiments, the effector cell is a T cell. In some embodiments, the effector cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the effector cell is modified to block or reduce expression of one or both of the endogenous TCR subunits from which the chimeric receptor polypeptide is derived. Modification of cells to disrupt gene expression includes any such technique known in the art, including, for example, RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR or TALEN-based gene knockout), and the like.

[0147] Methods for producing anti-LAB antibody moieties and constructs thereof In one aspect, the present application provides methods for making anti-LAB antibody moieties and constructs thereof.

[0148] Monoclonal antibodies In some embodiments, the anti-LAB antibody portion or construct thereof comprises a monoclonal antibody, which can be prepared, for example, using the hybridoma method as described by Kohler and Milstein, Nature, 256, 1975 and Sergeeva et al., Blood, 117(16):4262-4272, using the phage display method described herein and in the Examples below, or using recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567).

[0149] In the hybridoma method, a hamster, mouse, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent may include a polypeptide or a fusion protein of the protein of interest, or a complex containing at least two molecules. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See, for example, Goding, Monoclonal Antibodies: Principles and Practice (New York: Academic Press, 1986). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells are deficient in the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevents growth of HGPRT-deficient cells.

[0150] In some embodiments, the immortalized cell line fuses efficiently, supports stable high-level expression of antibody by selected antibody-producing cells, and is sensitive to a medium such as HAT medium. In some embodiments, the immortalized cell line is a mouse myeloma line, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, Calif., and the American Type Culture Collection, Manassas, Va. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al. Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc.: New York, 1987) pp. 51-63.

[0151] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0152] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Goding, supra. Suitable media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0153] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0154] Anti-LAB antibody moieties can also be identified by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0155] In a particular phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages, as described by Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages usually display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and also self antigens without any immunization, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0156] Antibodies or antigen-binding fragments thereof can be prepared using phage display to screen libraries for antibodies specific to Labyrinthin or a portion thereof. The libraries contain at least 1×10 9 (At least 1x10 9, 2.5x10 9 , 5x10 9 , 7.5x10 9 , 1x10 10 , 2.5x10 10 , 5x10 10 , 7.5x10 10 , or 1x10 11 The library may be a human scFv phage display library having a diversity of unique human antibody fragments, such as any of the following: (a) a human scFv phage display library having a diversity of unique human antibody fragments, such as any of the following: (i ... In some embodiments, the heavy chain CDR3 of the semi-synthetic human library has a length of about 5 to about 24 (e.g., about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids. In some embodiments, the library is a non-human phage display library.

[0157] Phage clones that bind with high affinity to labyrinthin or a portion thereof can be selected by repeatedly binding phages to labyrinthin bound to a solid support (such as beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phages and elution of specifically bound phages. In one example of solution panning, labyrinthin can be biotinylated and immobilized on a solid support. Biotinylated labyrinthin is mixed with a phage library and a solid support such as streptavidin-conjugated Dynabeads M-280, and then labyrinthin-phage-bead complexes are isolated. Bound phage clones are then eluted and used to infect suitable host cells such as E. coli XL1-Blue for expression and purification. Panning can be performed in multiple (such as about any of 2, 3, 4, 5, 6 or more) rounds using solution panning, cell panning, or a combination of both to enrich for phage clones that specifically bind to labyrinthin. The enriched phage clones can be tested for specific binding to Labyrinthin by any method known in the art, including, for example, ELISA and FACS.

[0158] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a murine antibody). The hybridoma cells described above or the Labyrinthin-specific phage clones of the invention can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains and / or framework regions for homologous non-human sequences (U.S. Pat. No. 4,816,567; Morrison et al., supra), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention and can also be substituted for the variable domains of one antigen-binding site of an antibody of the invention to generate a chimeric bivalent antibody.

[0159] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, one method includes recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at any point in the Fc region to prevent cross-linking of the heavy chain. Alternatively, the relevant cysteine ​​residue is replaced with another amino acid residue or deleted to prevent cross-linking.

[0160] In vitro methods are also suitable for the preparation of monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using any method known in the art.

[0161] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusions are preferably with immunoglobulin heavy chain constant domains, containing at least part of the hinge, CH2 and CH3 regions. In some embodiments, the first heavy chain constant region (CH1), containing the site necessary for light chain binding, is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on generating bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology 121:210 (1986).

[0162] Human and Humanized Antibodies In some embodiments, the anti-LAB antibody portion or construct thereof comprises a humanized or human antibody. Humanized forms of non-human (e.g., murine) antibodies include chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (Fv, Fab, Fab', F(ab')). 2Humanized antibodies are human immunoglobulins (such as Fvs, scFvs, or other antigen-binding subsequences of antibodies) that typically contain minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the recipient's CDRs are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin consensus sequences. In some embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0163] Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Such non-human amino acid residues are often referred to as "import" residues, and typically come from an "import" variable domain. In some embodiments, humanization can be essentially performed by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)). Such "humanized" antibodies are thus antibodies in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species (U.S. Pat. No. 4,816,567). In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0164] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that, upon immunization, can produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807; and WO 97 / 17852. Alternatively, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and in Marks et al., Bio / Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

[0165] Human antibodies can be produced by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275) or by 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). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991).

[0166] multispecific antibodies In some embodiments, the anti-LAB antibody moiety or construct thereof comprises a multispecific antibody. Suitable methods for producing multispecific (e.g., bispecific) antibodies are well known in the art. For example, the production of bispecific antibodies can be based on the co-expression of two immunoglobulin heavy / light chain pairs, each of which has a different specificity and which, upon association, results in a heterodimeric antibody (see, for example, Milstein and Cuello, Nature, 305: 537-539 (1983); WO 93 / 08829, and Traunecker et al., EMBO J.10: 3655 (1991)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) generate a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually performed by an affinity chromatography step. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO, 10: 3655-3659 (1991). Alternatively, heavy and light chain combinations can be guided by utilizing species-restricted pairings (see, e.g., Lindhofer et al., J. Immunol., 155:219-225 (1995)), and heavy chain pairings can be guided by using "knobs-into-holes" engineering of CH3 domains (see, e.g., U.S. Pat. No. 5,731,168; Ridgway et al., Protein Eng., 9(7):617-621 (1996)). Multispecific antibodies can also be generated by manipulating electrostatic steering effects to generate antibody Fc heterodimeric molecules (see, e.g., WO 2009 / 089004 (A1)). In yet another method, stable bispecific antibodies can be generated by controlled Fab arm exchange, in which two parent antibodies with point mutations in the CH3 domains that match different antigen specificities are mixed under reducing conditions, dissociated, reconstituted, and reoxidized to form a highly pure bispecific antibody.Labrigin et al., Proc. Natl. Acad. Sci., 110(13):5145-5150 (2013). Such antibodies comprising a mixture of heavy / light chain pairs are also referred to herein as "heteromultimeric antibodies."

[0167] Antibodies or antigen-binding fragments thereof with different specificities can also be chemically crosslinked to generate multispecific heteroconjugate antibodies. For example, two F(ab')2 molecules, each with specificity for a different antigen, can be chemically linked. Pullarkat et al., Trends Biotechnol., 48:9-21 (1999). Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for the treatment of HIV infection. WO 91 / 00360; WO 92 / 200373; EP 03089. It is contemplated that antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking 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, as well as those disclosed in US Pat. No. 4,676,980.

[0168] In some embodiments, multispecific antibodies can be prepared using recombinant DNA technology. For example, bispecific antibodies can be engineered by fusing two scFvs, such as by fusing two scFvs via a peptide linker, resulting in a tandem scFv. One example of a tandem scFv is a bispecific T cell engager. A bispecific T cell engager is made by linking an anti-CD3 scFv to an scFv specific for a surface antigen of a target cell, such as a tumor-associated antigen (TAA), resulting in the redirection of T cells to the target cell. Mack et al., Proc. Natl. Acad. Sci., 92:7021-7025 (1995); Brischwein et al., Mol. Immunol., 43(8):1129-1143 (2006). By shortening the length of the peptide linker between the two variable domains, they can be prevented from self-assembly and forced to pair with the domains on a second polypeptide, resulting in compact bispecific antibodies called diabodies (Dbs). Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448 (1993). The two polypeptides of a Db each contain a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one polypeptide are forced to pair with the complementary VL and VH domains of another polypeptide, thereby forming two antigen-binding sites. In a modification of this format, the two polypeptides are linked by another peptide linker, resulting in single-chain diabodies (scDbs). Yet another modification of the Db format allows for the generation of dual affinity retargeting (DART) bispecific antibodies by introducing a disulfide bond between the C-terminal cysteine ​​residues of each polypeptide, optionally including a domain before the C-terminal cysteine ​​residue that drives assembly of the desired heterodimeric structure. Veri et al., Arthritis Rheum., 62(7):1933-1943 (2010).Dual variable domain immunoglobulins (DVD-Ig) in which the target-binding variable domains of two monoclonal antibodies are combined via a naturally occurring linker to generate tetravalent bispecific antibodies. TM ) are also known in the art. Gu and Ghayur, Methods Enzymol., 502:25-41 (2012). Yet another format, Dock-and-Lock (DNL), utilizes dimerization of a peptide (DDD2) derived from the regulatory subunit of human cAMP-dependent protein kinase (PKA) with a peptide (AD2) derived from the anchor domain of human A-kinase anchor protein (AKAP) to prepare bispecific antibodies. Rossi et al., Proc. Natl. Acad. Sci., 103:6841-6846 (2006).

[0169] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been made using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). This method can also be used to make antibody homodimers.

[0170] Anti-LAB antibody partial variants In some embodiments, amino acid sequence variants of the antibody moieties provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody moiety. Amino acid sequence variants of the antibody moiety can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody moiety or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody moiety. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0171] In some embodiments, antibody portion variants are provided that have one or more amino acid substitutions. Target sites for substitution mutagenesis include HVRs and FRs. Amino acid substitutions can be introduced into the antibody portion of interest, and the products can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0172] Conservative substitutions are shown in Table 4 below. TIFF2025060688000005.tif182170

[0173] Amino acids can be divided into different classes based on the properties of their common side chains: a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues that affect chain orientation: Gly, Pro; f. Aromatic: Trp, Tyr, Phe

[0174] Non-conservative substitutions entail exchanging a member of one of these classes for a member of another class.

[0175] An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated, and the variant antibody moieties are displayed on phage and screened for a particular biological activity (e.g., binding affinity). Modifications (e.g., substitutions) may be made in the HVRs, for example, to improve antibody moiety affinity. Such modifications are made in HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or specificity determining residues (SDRs), and the resulting mutant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).

[0176] In some embodiments 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 generated. The library is then screened to identify any antibody partial variants with the desired affinity. Another method of introducing diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0177] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the antigen-binding ability of the antibody moiety. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such modifications may be outside of HVR "hot spots" or SDRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains only one, two, or three amino acid substitutions.

[0178] A useful method for identifying residues or regions of an antibody moiety that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody moiety with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of the antigen-antibody moiety complex can be determined to identify contact points between the antibody moiety and the antigen. Such contact and adjacent residues can be targeted or removed as candidates for substitution. The variants can be screened to determine whether they contain the desired properties.

[0179] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody moiety with an N-terminal methionyl residue. Other insertional variants of the antibody moiety include the fusion to the N- or C-terminus of the antibody moiety to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody moiety.

[0180] Fc region variants In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the full-length anti-LAB antibody portion provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced antibody-dependent cellular cytotoxicity (ADCC) effector function, often in conjunction with binding to Fc receptors (FcRs). In some embodiments, the Fc region variant has reduced ADCC effector function. There are many examples of changes or mutations to the Fc sequence that can alter effector function. For example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001) describe antibody variants with improved or reduced binding to FcRs. The contents of these publications are specifically incorporated herein by reference.

[0181] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., cancer cells) whose membrane surface antigens are bound by a specific antibody (e.g., an anti-LAB antibody). A typical ADCC involves the activation of NK cells by antibodies. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibodies bound to the surface of target cells. The most common Fc receptor on the surface of NK cells is called CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of an antibody activates the NK cell and releases cytolytic granules, which results in apoptosis of the target cell. The contribution of ADCC to tumor cell killing can be measured in a specific test using NK-92 cells transfected with a high affinity FcR. The results are compared to wild-type NK-92 cells that do not express an FcR.

[0182] In some embodiments, the present invention contemplates anti-LAB construct variants that contain an F-C region that retains some, but not all, effector functions, making them desirable candidates for applications in which the half-life of the anti-LAB construct in vivo is important, but certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat' Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay procedures may be employed (e.g., ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96 TMNon-radioactive cytotoxicity assays (see Promega, Madison, Wis.) Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat' Acad. Sci. USA 95:652-656 (1998). C1q binding assays may be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may 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, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life measurements 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)).

[0183] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0184] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0185] In some embodiments, anti-LAB constructs (e.g., full-length anti-LAB antibodies) variants are provided that comprise a variant Fc region that comprises one or more amino acid substitutions that improve ADCC. In some embodiments, the variant Fc region comprises one or more amino acid substitutions that improve ADCC, where the substitutions are at positions 298, 333, and / or 334 (EU numbering of residues) of the variant Fc region. In some embodiments, the anti-LAB constructs (e.g., full-length anti-LAB antibodies) variants comprise the following amino acid substitutions in their variant Fc region: S298A, E333A, and K334A.

[0186] In some embodiments, modifications are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0187] In some embodiments, variants of anti-LAB constructs (e.g., full-length anti-LAB antibodies) are provided that include variant Fc regions that include one or more amino acid substitutions that increase half-life and / or improve binding to neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US Patent Application Publication No. 2005 / 0014934 (A1) (Hinton et al.). These antibodies include Fc regions with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of 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, such as a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0188] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:73840 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351.

[0189] Anti-LAB constructs (such as full-length anti-LAB antibodies) comprising any of the Fc variants described herein, or combinations thereof, are contemplated.

[0190] Glycosylation variants In some embodiments, the anti-LAB constructs provided herein are modified to increase or decrease the extent to which the anti-LAB construct is glycosylated. Addition or deletion of glycosylation sites to an anti-LAB construct can be conveniently accomplished by modifying the amino acid sequence of the anti-LAB construct or its polypeptide portion such that one or more glycosylation sites are created or removed.

[0191] When the anti-LAB construct comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the anti-LAB construct of the present invention may be performed to generate anti-LAB construct variants with specific improved properties.

[0192] Anti-LAB constructs (such as full-length anti-LAB antibodies) variants comprising an Fc region are provided in which the carbohydrate structures attached to the Fc region have reduced fucose or are devoid of fucose, which may improve ADCC function. Specifically, anti-LAB constructs are contemplated herein that have reduced fucose relative to the amount of fucose on the same anti-LAB construct produced in wild-type CHO cells. That is, they are characterized by having a lower amount of fucose when produced by native CHO cells (e.g., CHO cells that produce a native glycosylation pattern, such as CHO cells that contain a native FUT8 gene) than they would otherwise. In some embodiments, the anti-LAB constructs have less than about 50%, 40%, 30%, 20%, 10%, or 5% of their N-linked glycans containing fucose. For example, the amount of fucose in such anti-LAB constructs may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In some embodiments, the anti-LAB construct does not contain any of its N-linked glycans containing fucose, i.e., the anti-LAB construct is completely fucose-free, fucose-free, or afucosylated. The amount of fucose is determined by calculating the average amount of fucose in the glycan of Asn297 relative to the sum of all glycans (complex, hybrid, high mannose structures, etc.) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 of the Fc region (Eu numbering of Fc region residues), although due to minor sequence variations in antibodies, Asn297 may be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, U.S. Patent Application Publication No. 2003 / 0115614, U.S. Patent Application Publication No. 2002 / 0164328, U.S. Patent Application Publication No. 2004 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, and the like. No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO 2005 / 053742, WO 2002 / 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, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); Presta, L., U.S. Patent Application Publication No. 2003 / 0157108; and Adams et al., WO 2004 / 056312 (especially 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).

[0193] The variant of the anti-LAB construct (such as a full-length anti-LAB antibody) further comprises a bisected oligosaccharide, for example, the biantennary oligosaccharide attached to the Fc region of the anti-LAB construct is bisected by GlcNAc. Such variant of the anti-LAB construct (such as a full-length anti-LAB antibody) may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairett et al.); U.S. Patent No. 6,602,684 (Umana et al.); U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851861 (2006). Also provided is a variant of the anti-LAB construct (such as a full-length anti-LAB antibody) having at least one galactose residue in the oligosaccharide attached to the Fc region. Such anti-LAB construct variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0194] In some embodiments, the anti-LAB construct (such as a full-length anti-LAB antibody) variants comprising an Fc region can bind to FcγRIII. In some embodiments, the anti-LAB construct (such as a full-length anti-LAB antibody) variants comprising an Fc region have ADCC activity in the presence of human effector cells or have increased ADCC activity in the presence of human effector cells compared to an otherwise identical anti-LAB construct (such as a full-length anti-LAB antibody) comprising a human wild-type IgG1 Fc region.

[0195] Cysteine ​​engineered variants In some embodiments, it may be desirable to generate cysteine ​​engineered anti-LAB constructs (such as full-length anti-LAB antibodies) in which one or more amino acid residues are replaced with cysteine ​​residues. In some embodiments, the replacement residues occur at accessible sites of the anti-LAB construct. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the anti-LAB construct, which can be used to conjugate the anti-LAB construct to other moieties, such as drug moieties or linker-drug moieties, to generate anti-LAB immunoconjugates, as further described herein. Cysteine ​​engineered anti-LAB constructs (such as full-length anti-LAB antibodies) can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0196] derivative In some embodiments, the anti-LAB constructs provided herein may be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of anti-LAB constructs include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxolane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the anti-LAB construct may vary, and when multiple polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the anti-LAB construct to be improved, whether the anti-LAB construct derivative will be used in treatment under defined conditions, etc.

[0197] In some embodiments, a conjugate is provided between an anti-LAB construct and a non-proteinaceous moiety that can be selectively heated by exposure to radiation.In some embodiments, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)).The radiation can be of any wavelength, including but not limited to wavelengths that do not harm normal cells, but heat the non-proteinaceous moiety to a temperature that kills cells in the vicinity of the anti-LAB construct-non-proteinaceous moiety.

[0198] Anti-LAB CAR, anti-LAB TCR, and anti-LAB bispecific T cell engager In one aspect, the present application provides an anti-LAB construct, where the anti-LAB construct is, for example, an anti-LAB CAR, an anti-LAB TCR, or an anti-LAB bispecific T cell engager. Methods for producing such anti-LAB constructs are known in the art and are, for example, described in the above sections.

[0199] Preparation of effector cells The invention in one aspect provides, for example, effector cells (e.g., lymphocytes, e.g., T cells) that express an anti-LAB CAR. Provided herein are exemplary methods of preparing effector cells (such as T cells) that express an anti-LAB CAR (anti-LAB CAR effector cells, such as anti-LAB CAR T cells).

[0200] In some embodiments, anti-LAB CAR effector cells (such as T cells) can be generated by introducing a vector (such as a lentiviral vector) comprising an anti-LAB CAR (e.g., a CAR comprising an anti-LAB antibody portion and CD28 and CD3 zeta intracellular signaling sequences) into an effector cell (such as a T cell). In some embodiments, the anti-LAB CAR effector cells (such as T cells) of the present invention can replicate in vivo, resulting in long-term persistence that can lead to sustained control of labyrinthin-positive disease (cancer, such as adenocarcinoma).

[0201] In some embodiments, the present invention relates to administering genetically modified T cells expressing anti-LAB CARs for the treatment of patients with or at risk of having Labyrinthin-positive disease using lymphocyte infusion. In some embodiments, autologous lymphocyte infusion is used in the treatment. Autologous PBMCs are collected from the patient in need of treatment, and T cells are activated and expanded using methods described herein and known in the art, and then infused back into the patient.

[0202] In some embodiments, the anti-LAB CAR T cells express an anti-LAB CAR comprising an anti-LAB antibody portion (also referred to herein as "anti-LAB CAR T cells"). In some embodiments, the anti-LAB CAR T cells express an anti-LAB CAR comprising an extracellular domain comprising an anti-LAB antibody portion and an intracellular domain comprising the intracellular signaling sequences of CD3ζ and CD28. The anti-LAB CAR T cells of the invention can undergo robust in vivo T cell proliferation and can establish high levels of labyrinthin-specific memory cells that persist for extended periods in the blood and bone marrow. In some embodiments, the anti-LAB CAR T cells of the invention infused into a patient can eliminate labyrinthin-presenting cells, such as labyrinthin-presenting cancer cells, in vivo in patients with labyrinthin-positive disease. In some embodiments, the anti-LAB CAR T cells of the invention infused into a patient can eliminate labyrinthin-presenting cells, such as labyrinthin-presenting cancer cells, in vivo in patients with labyrinthin-positive disease that is resistant to at least one conventional therapy.

[0203] Prior to the expansion and genetic modification of T cells, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, any number of T cell lines available in the art can be used. In some aspects of the invention, T cells are isolated by Ficoll filtration. TMCells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as cell separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for the next processing step. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium, may lack magnesium, and may lack many, if not all, divalent cations. As one of skill in the art will readily appreciate, the washing step can be accomplished by methods known to those of skill in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells are washed with Ca 2+ -Free, Mg 2+ The cells may be resuspended in a variety of biocompatible buffers, such as -free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0204] In some embodiments, the T cells are activated by lysing red blood cells and depleting monocytes, e.g., by PERCOLL TM It is isolated from peripheral blood lymphocytes by gradient centrifugation or counterflow centrifugal elutriation. + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO +Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3×28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. In some embodiments, the time period is about 30 minutes. In some embodiments, the time period ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In some embodiments, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. For isolating T cells from leukemia patients, longer incubation times, such as 24 hours, can be used to increase cell yield. In any situation where there are few T cells compared to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals, longer incubation times can be used to isolate T cells. Additionally, the use of longer incubation times can improve the ability to differentiate CD8 + The efficiency of T cell capture can be increased. Thus, by simply shortening or lengthening the time that T cells can bind to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells, subpopulations of T cells can be preferentially selected for at the beginning of culture or at other times during the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for at the beginning of culture or at other desired times. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In some embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to additional rounds of selection.

[0205] Enrichment of T cell populations by negative selection can be achieved with a combination of antibodies against surface markers unique to the negatively selected cells. One method is 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 by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, CD4 + , CD25 + , CD62Lhi, GITR + , and FoxP3 + Alternatively, in some embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar selection methods.

[0206] For isolation of the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and beads. For example, in some embodiments, a concentration of about 2 billion cells / ml is used. In some embodiments, a concentration of about 1 billion cells / ml is used. In some embodiments, more than about 100 million cells / ml is used. In some embodiments, a concentration of cells is used of about 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml. In some embodiments, a concentration of cells is used of about 75, 80, 85, 90, 95, or 100 million cells / ml. In some embodiments, a concentration of about 125 million or about 150 million cells / ml is used. Using a higher concentration can result in increased cell yield, cell activation, and cell proliferation. Furthermore, using a higher cell concentration allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or from samples where many tumor cells are present (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, using a higher concentration of cells can result in more efficient capture of CD8 T cells that normally have weak expression of CD28. + T cells can be selected more efficiently.

[0207] In some embodiments of the present invention, T cells are obtained directly from the patient after treatment. In this regard, it has been observed that after certain cancer treatments, particularly treatments with drugs that damage the immune system, the quality of the obtained T cells may be optimal or improved for their ability to expand ex vivo immediately after treatment during the period when the patient is usually recovering from the treatment. Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period. Furthermore, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and pre-transplantation treatments can be used to create conditions in the subject that favor repopulation, recirculation, regeneration, and / or expansion of certain cell types, particularly during a defined time frame after treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0208] Whether or not the T cells have been genetically modified to express a desired anti-LAB CAR, the T cells can generally be activated and expanded using methods described, for example, in 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.

[0209] In general, the T cells of the present invention are expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, the T cell population can be stimulated, for example, by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For costimulation of an accessory molecule on the surface of the T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. CD4 + T cells or CD8 + To stimulate the proliferation of T cells, they are contacted with anti-CD3 and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besançon, France), and can be used as well as other methods commonly 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).

[0210] Preparation of immunoconjugates Anti-LAB immunoconjugates can be prepared using any method known in the art, see, for example, WO 2009 / 067800, WO 2011 / 133886, and US Patent Publication No. 2014321229, which are incorporated by reference in their entireties.

[0211] The anti-LAB antibody portion of the anti-LAB immunoconjugate can be "attached" to the effector molecule by any means that allows the anti-LAB antibody portion to be bound or linked to the effector molecule. For example, the anti-LAB antibody portion of the anti-LAB immunoconjugate can be attached to the effector molecule by chemical or recombinant means. Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare the anti-LAB immunoconjugate. The method used to conjugate the anti-LAB antibody portion and the effector molecule must be capable of binding the binding protein to the effector molecule without interfering with the ability of the binding protein to bind to an antigen on a target cell.

[0212] The anti-LAB antibody portion of the anti-LAB immunoconjugate can be indirectly linked to an effector molecule.For example, the anti-LAB antibody portion of the anti-LAB immunoconjugate can be directly linked to a liposome that contains one of several types of effector molecules.Effector molecules and / or anti-LAB antibody portions can also be bound to solid surfaces.

[0213] In some embodiments, the anti-LAB antibody moiety and the effector molecule of the anti-LAB immunoconjugate are both proteins and can be conjugated using techniques well known in the art. There are hundreds of cross-linking agents available that can conjugate two proteins. (See, for example, "Chemistry of Protein Conjugation and Crosslinking" 1991, Shans Wong, CRC Press, Ann Arbor). The cross-linking agent is generally selected based on the reactive functional groups available or inserted on the anti-LAB antibody moiety and / or the effector molecule. Furthermore, photoactivatable cross-linking agents can be used in the absence of reactive groups. In certain instances, it may be desirable to include a spacer between the anti-LAB antibody moiety and the effector molecule. Cross-linking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyladipimidate and bis(diazobenzidine) and heterobifunctional agents: m-maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.

[0214] In some embodiments, the anti-LAB antibody portion of the anti-LAB immunoconjugate can be engineered with specific residues for chemical attachment of effector molecules. Specific residues used for chemical attachment of molecules known in the art include lysine and cysteine. Crosslinkers are inserted on the anti-LAB antibody portion and are selected based on the reactive functional groups available on the effector molecule.

[0215] Anti-LAB immunoconjugates can also be prepared using recombinant DNA technology.In such cases, the DNA sequence encoding the anti-LAB antibody portion is fused to the DNA sequence encoding the effector molecule, resulting in a chimeric DNA molecule.This chimeric DNA sequence is transfected into a host cell that expresses the fusion protein.The fusion protein can be recovered from cell culture and purified using techniques known in the art.

[0216] Examples of methods for attaching a label effector molecule to a binding protein include those described in Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth.40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Radioimmunoimaging And Radioimmunotherapy, Elsevier, NY (1983); and Colcher et al., "Use Of Monoclonal Antibodies As Radiopharmaceuticals For The Localization Of Human Carcinoma Xenografts In Athymic Mice", Meth. Enzymol., 121:802-16 (1986).

[0217] Radiolabels or other labels can be incorporated into the immunoconjugate in known manner, for example, the peptides can be biosynthesized or synthesized by chemical amino acid synthesis using appropriate amino acid precursors that include, for example, fluorine-19 instead of hydrogen. 99 Tc or 123 I, 186 Re, 188 Re, 111 Labels such as In can be attached through cysteine ​​residues of the peptide. Yttrium-90 can be attached through lysine residues. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun. 80:49-57 (1978)). Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0218] Immunoconjugates of antibody moieties and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and diactive fluorine compounds (e.g., 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 chelating agent for conjugation of radionucleotides to antibodies. See, for example, WO 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic agent inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0219] The anti-LAB immunoconjugates of the present invention expressly contemplate ADCs prepared with, but not limited to, the following commercially available crosslinkers (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA): 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-vinylsulfone)benzoate. See pages 467-498 of the 2003-2004 Applications Handbook and Catalog.

[0220] Pharmaceutical Compositions Also provided herein are compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising anti-LAB constructs. In some embodiments, the compositions further comprise cells (e.g., effector cells, e.g., T cells) associated with the anti-LAB construct. In some embodiments, pharmaceutical compositions are provided comprising the anti-LAB construct and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical compositions further comprise cells (e.g., effector cells, e.g., T cells) associated with the anti-LAB construct.

[0221] Suitable preparations of anti-LAB constructs are obtained in the form of lyophilized preparations or aqueous solutions by mixing anti-LAB constructs having the desired purity with optional pharma- ceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or TWEEN. TM , PLURONICS TM or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary formulations are described in WO 98 / 56418, which is expressly incorporated herein by reference. Lyophilized formulations adapted for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation may be administered subcutaneously to the individual treated herein. Lipofectin or liposomes may be used to deliver the anti-LAB constructs of the present invention to cells.

[0222] The formulations herein may also contain one or more active compounds, preferably with complementary activities that do not adversely affect each other, in addition to the anti-LAB constructs necessary for the particular indication being treated. For example, it may be desirable to provide an anti-tumor agent, growth inhibitory agent, cytotoxic agent or chemotherapeutic agent in addition to the anti-LAB constructs. Such molecules are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents will depend on the amount of anti-LAB constructs present in the formulation, the type of disease or disorder or treatment, and other factors mentioned above. They are generally used in the same dosages and routes of administration as described herein, or about 1 to 99% of the dosages previously used.

[0223] The anti-LAB constructs may also be encapsulated in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Sustained release formulations may also be prepared.

[0224] A sustained release formulation of the anti-LAB construct can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody (or fragments thereof), which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT TM(Injectable microspheres made of lactic acid-glycolic acid copolymer and leuprolide acetate) and degradable lactic acid-glycolic acid copolymers, such as poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, while certain hydrogels release proteins for shorter times. If encapsulated antibodies remain in the body for a long period of time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and altered immunogenicity. Depending on the mechanism involved, rational strategies can be devised for the stabilization of anti-LAB constructs. For example, if the aggregation mechanism is found to be intermolecular SS bond formation by thio-disulfide exchange, stabilization can be achieved by modification of sulfhydryl residues, lyophilization from acidic solution, control of water content, use of appropriate additives, and development of specific polymer matrix compositions.

[0225] In some embodiments, the anti-LAB construct is formulated in a buffer comprising citrate, NaCl, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination of the above. In some embodiments, the anti-LAB construct is formulated in a buffer comprising about 100 mM to about 150 mM glycine. In some embodiments, the anti-LAB construct is formulated in a buffer comprising about 50 mM to about 100 mM NaCl. In some embodiments, the anti-LAB construct is formulated in a buffer comprising about 10 mM to about 50 mM acetate. In some embodiments, the anti-LAB construct is formulated in a buffer comprising about 10 mM to about 50 mM succinate. In some embodiments, the anti-LAB construct is formulated in a buffer comprising about 0.005% to about 0.02% polysorbate 80. In some embodiments, the anti-LAB construct is formulated in a buffer having a pH between about 5.1 and 5.6. In some embodiments, the anti-LAB construct is formulated in a buffer containing 10 mM citrate, 100 mM NaCl, 100 mM glycine, and 0.01% polysorbate 80, where the formulation has a pH of 5.5. The formulation used for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through a sterile filtration membrane.

[0226] How to use the anti-LAB construct The applicable anti-LAB constructs and / or compositions can be administered to an individual (e.g., a mammal, such as a human) to treat and / or prevent diseases and / or disorders, including, for example, cancer (such as adenocarcinoma). In some embodiments, the cancer is a labyrinthin-positive cancer. In some embodiments, the cancer is an adenocarcinoma, such as a labyrinthin-positive adenocarcinoma. Thus, the present application provides a method of treating a labyrinthin-positive disease (such as cancer) in an individual, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising an anti-LAB construct comprising an anti-LAB antibody moiety, such as any one of the anti-LAB constructs described herein. In some embodiments, the composition comprises a cell (such as an effector cell) expressing an anti-LAB construct (such as a CAR).

[0227] For example, in some embodiments, a method for treating a labyrinthin-positive disease in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an anti-LAB construct comprising an anti-LAB antibody portion that specifically binds to labyrinthin. In some embodiments, a method for treating a labyrinthin-positive disease in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an effector cell (e.g., a T cell) expressing an anti-LAB construct (e.g., an anti-LAB CAR or anti-LAB TCR as described herein). In some embodiments, an individual is selected for treatment if a cancer sample from the individual shows that at least about 10% or more of the cancerous cells in the sample are positive for labyrinthin, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95%, as determined using immunohistochemistry (IHC) techniques. In some embodiments, an individual is selected for treatment if a cancer sample from the individual shows that about 10% or more, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95%, of a subpopulation of cancerous cells in the sample, such as cells in the periphery of a tumor, are positive for Labyrinthin as determined using immunohistochemistry (IHC) techniques.

[0228] In some embodiments, the individual is a human.

[0229] The dose of the anti-LAB construct composition administered to an individual (such as a human) may vary depending on the particular composition, the mode of administration, and the type of disease being treated. In some embodiments, the amount of the composition is effective to produce an objective response (such as partial or complete remission). In some embodiments, the amount of the anti-LAB construct (e.g., full-length anti-LAB antibody, multispecific anti-LAB molecule, or anti-LAB immunoconjugate) in the composition is, for example, in the range of about 0.001 μg to about 1000 μg. In some embodiments of any of the above aspects, the effective amount of the anti-LAB construct (e.g., full-length anti-LAB antibody, multispecific anti-LAB molecule, or anti-LAB immunoconjugate) in the composition is in the range of about 0.1 μg / kg to about 100 mg / kg of total body weight.

[0230] The anti-LAB construct composition can be administered to an individual (such as a human) via a variety of routes, including, for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intratracheal, subcutaneous, intradermal, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, sustained release formulations of the composition can be used.

[0231] The present application also provides methods for redirecting the specificity of effector cells (immune cells, such as T cells) to cells expressing Labyrinthin using anti-LAB CARs or anti-LAB TCRs. Thus, the present application also provides a method of stimulating an effector cell-mediated response (such as a T cell-mediated immune response) against a target cell population or tissue expressing Labyrinthin in a mammal, comprising administering to the mammal an effector cell (such as a T cell) expressing an anti-LAB CAR and an anti-LAB TCR.

[0232] Anti-LAB CARs or anti-LAB TCR effector cells (such as T cells) expressing anti-LAB CARs can be infused into a recipient in need thereof. The infused cells can kill the recipient's labyrinthin-expressing cells. In some embodiments, unlike antibody therapy, anti-LAB CARs or anti-LAB TCR effector cells (such as T cells) can replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0233] Ex vivo procedures are well known in the art and are discussed in more detail below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing an anti-LAB CAR or anti-LAB TCR as disclosed herein. The anti-LAB CART or anti-LAB TCRT cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the anti-LAB CART or anti-LAB TCRT cells can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

[0234] In addition to using cell-based vaccines in terms of ex vivo immunization, the present application also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

[0235] The exact amount of the anti-LAB CAR or anti-LAB TCR effector cell (e.g., T cell) composition of the present application to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in condition of the patient (subject). In some embodiments, a pharmaceutical composition comprising an anti-LAB CAR or anti-LAB TCR effector cell (e.g., T cell) is administered in an amount of about 10 4 About 10 9The cells are administered in a dosage of cells / kg body weight. Anti-LAB CAR or anti-LAB TCR effector cell (such as T cell) compositions may also be administered multiple times in these dosages. The cells may be administered using injection techniques commonly known in immunotherapy. The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0236] Administration of anti-LAB CAR or anti-LAB TCR effector cells (such as T cells) can be performed in any convenient manner, including aerosol inhalation, injection, oral ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intralymph node, intramedullary, intramuscularly, intravenously (iv) or intraperitoneally. In some embodiments, the anti-LAB CAR or anti-LAB TCR effector cell (such as T cells) compositions of the present application are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the anti-LAB CAR or anti-LAB TCR effector cell (such as T cells) compositions of the present application are administered by intravenous injection. The anti-LAB CAR or anti-LAB TCR effector cell (such as T cells) compositions can be directly injected into a tumor, lymph node, or site of infection.

[0237] The methods disclosed herein may include administering to an individual multiple doses of the anti-LAB constructs and / or compositions described herein over a period of time. In some embodiments, the frequency and / or dosage of the anti-LAB constructs and / or compositions described herein is adjusted over the course of treatment based on the judgment of the administering physician.

[0238] The methods for treatment and / or prevention disclosed herein are useful for treating or preventing a proliferative disease, such as cancer, in an individual. In some embodiments, the cancer is a labyrinthin-expressing cancer, such as a labyrinthin-positive cancer. In some embodiments, the cancer is an adenocarcinoma. In some embodiments, the cancer is an early stage cancer, a non-metastatic cancer, a primary cancer, an advanced cancer, a locally advanced cancer, a metastatic cancer, a cancer in remission, a recurrent cancer, a resistant cancer, or a refractory cancer. In some embodiments, the cancer is a localized resectable cancer (e.g., a tumor that is confined to a part of an organ that allows complete surgical removal), a localized unresectable cancer (e.g., a localized tumor that is unresectable due to important vasculature), or an unresectable cancer. In some embodiments, the cancer is a stage I tumor, a stage II tumor, a stage III tumor, a stage IV tumor, an N1 tumor, or an M1 tumor according to the TNM classification.

[0239] The methods disclosed herein are useful for treating or preventing cancer, such as Labyrinthin-expressing cancer, in an individual. In some embodiments, the individual has one or more of the following characteristics: (i) the ability to understand and willingness to sign an informed consent form; (ii) at least 18 years old with histologically confirmed adenocarcinoma and / or Labyrinthin-expressing cancer; (iii) previously treated with at least one systemic therapy (chemotherapy and / or biotherapy) and either did not respond / progressed during treatment, progressed after completion of systemic therapy, or refused all other treatments; (iv) the tumor overexpresses Labyrinthin antigen, as determined by immunohistochemical evaluation screening of paraffin-embedded archival specimens, at an intensity of at least 2× background according to scoring by a single reference pathologist; (v) any number of prior chemotherapy regimens; (vi) documentation of a delayed-type hypersensitivity (DTH) response to a common recall antigen prior to the first vaccine injection; (vii) a performance status of ≥60% on the Karnofsky scale; (viii) life expectancy at time of treatment ≥6 months; (ix) measurable or evaluable disease; (x) a pretreatment absolute granulocyte count (AGC) ≥1000 and a pretreatment platelet count ≥75000 obtained within 4 weeks prior to the first vaccine injection; (xi) a pretreatment serum creatinine of ≤1.5 mg / dl is required; and (xii) serum bilirubin ≤1.5 and AST ≤2.5 × institutional upper limit of normal (≤5 × for liver metastases).

[0240] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the disclosure of this application. The disclosure is further illustrated by the following examples, which should not be construed as limiting the disclosure in scope or spirit to the specific procedures described therein. EXAMPLES

[0241] Example 1 This example shows the development and testing of functional single chain variable fragments (scFv) and antigen binding fragments (Fab) derived from an anti-Labyrinthin antibody (MCA 44-3A6).

[0242] The heavy chain variable domain (VH) and light chain variable domain (VH) cDNA of MCA 44-3A6 were cloned with oligo primers suitable for cDNA synthesis and PCR. The cloning was based on the sequences published in the immunoglobulin database. Briefly, approximately 5x10 8 RNA prepared from the hybridoma cells was used for the preparation of cDNA using commercially available reagents. The amplified cDNAs of VH and VL were digested with restriction enzymes at the sites placed during PCR amplification. The genes were then subcloned into a phage display vector to construct scFvs for library selection purposes.

[0243] The library of scFvs was then subcloned. For each scFV, its sequence was fused so that upon subsequent expression the product would have a C-terminal myc tag. For selection of the phage library, protein antigens (histidine-tagged labyrinthin) were immobilized overnight on 96-well plates. Aliquots of library phages were placed into assigned wells. Bound phages were eluted and added to TG1 culture medium for infection and rescued by KO7 helper phage. Infected TG1 cells were grown (overnight at 30°C) and bacterial culture supernatants were harvested, precipitated, and resuspended in PBS for subsequent selection or phage ELISA.

[0244] For antigen preparation, the extracellular domain of Labyrinthin was cloned and expressed as a GST fusion protein using pGEX-2T. Crude preparations were easily prepared using size-exclusion chromatography, and pure preparations were made by affinity chromatography. Due to the presence of negatively charged acidic residues in some parts of the protein, Labyrinthin was observed to strongly aggregate as purification proceeded. Therefore, a histidine-tagged modified Labyrinthin protein (the very hydrophobic first 22 amino acids: leader-like sequence excluded) was generated and used in the antigen-antibody screening procedure (phage display) described above. Although histidine is not expected to have a significant effect on the secondary structure of Labyrinthin, given the issues surrounding the purification of this protein, an alternative KLH-conjugated peptide corresponding to amino acids #109-130, which contains the known binding site of MCA 44-3A6, was also synthesized. Thus, the modified His-Labyrinthin was used in the phage-display antibody selection, but the synthetic peptide was also included in the Biacore binding measurements to ensure the selection of the best antibody candidates.

[0245] For the phage ELISA, His-Labyrinthin was coated onto ELISA plates (overnight at 4°C). An aliquot of each phage to be tested was added to each well (incubated for 30–90 min at 37°C) and washed extensively with PBS. Phage-bound antigen was detected by incubation with HRP-conjugated anti-M13 antibody.

[0246] Purified protein labyrinthin antigens (His-labyrinthin and synthetic peptides) were then immobilized on a CM5 chip by amine coupling for Biacore measurements. Purified antibody lead variants (selected from a phage library) were injected into the system at various concentrations to evaluate the association and dissociation of the antibody-antigen interaction. Antibody affinity measurements were performed on a Biacore1000 instrument according to the manufacturer's protocol (Biacore, Inc., Piscataway, NJ). Depending on how close the affinities measured for different clones were to each other, the top three candidates were kept for final screening and propagation, and the highest affinity candidate was further evaluated using in vitro screening.

[0247] As shown in Figures 1A and 1B, initial binding assays were performed using His-tagged labyrinthin with the cloned and expressed protein (clone X373 scFv; Figure 1B) and the parental MCA 44-3A6 antibody (Figure 1A). X373 scFv was determined to have a dissociation constant (Kd) approximately four-fold lower than the parental MCA 44-3A6 antibody. Specifically, the Kd of X373scFv was 4.8x10 -7 M, and the Kd of MCA44-3A6 is 1.17x10 -7 It was measured as M.

[0248] The above development strategy was modified to generate an additional library for the Fab format. A Fab, X509Fab, was identified by screening and then evaluated for affinity to His-Labyrinthin and KLH-conjugated Labyrinthin-derived peptides using the Biacore method described above. The affinity measurements for X509Fab are reported in Table 5.

[0249] TIFF2025060688000006.tif28170

[0250] X509Fab was then tested for its ability to recognize native labyrinthin in adenocarcinoma cells. Serial sections (~6 μm thick) of A549 xenograft tumor sections were used to compare X509Fab with the parental MCA44-3A6 antibody under nearly identical conditions. Tissue sections exposed to 10 μg / ml of antibody (or no primary antibody) were then developed with the appropriate secondary antibody (HRP-conjugated goat anti-mouse or anti-human IgG) and DAB using a standard ABC method (VectorLaboratory, Burlingame, CA). Results (not shown) demonstrated that X509Fab binds to native labyrinthin and is at least equivalent to the parental antibody in its ability to recognize the epitope (labyrinthin) on cancer cells.

[0251] To determine whether the positive cell binding results of X509Fab represent specific recognition of labyrinthin, standard Western immunoblotting was performed with WI39 normal human fibroblast (negative control) and A549 lung adenocarcinoma cell (positive control) lysates. A clear band was observed at the expected size for labyrinthin (~40 kD), whereas no signal was obtained in the negative control (Figure 2). Similar to previous results with MCA 44-3A6, a faint lower band (degradation product) and a higher band (~80 kD) were observed, confirming that labyrinthin self-aggregates (Figure 2).

[0252] A summary of affinity measurements for scFv and Fab derived from anti-Labyrinthin antibody (MCA 44-3A6) against His-Labyrinthin and KLH-conjugated Labyrinthin-derived peptides is shown in Table 6.

[0253] TIFF2025060688000007.tif34170 (Sequence table) SEQUENCE LISTING <110> LABYRX IMMUNOLOGIC THERAPEUTICS (USA) LIMITED <120> CONSTRUCTS TARGETING LABYRINTHIN OR A PORTION THEREOF AND USES THEREOF <130> 18572-20002.40 <140> Not Yet Assigned <141> Concurrently Herewith <150> US 62 / 789,871 <151> 2019-01-08 <160> 38 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 255 <212> PRT <213> Homo sapiens <400> 1 Met Val Ile Ala Leu Leu Gly Val Trp Thr Ser Val Ala Val Val Trp 1 5 10 15 Phe Asp Leu Val Asp Tyr Glu Glu Val Leu Gly Lys Leu Gly Ile Tyr 20 25 30 Asp Ala Asp Gly Asp Gly Asp Phe Asp Val Asp Asp Ala Lys Val Leu 35 40 45 Leu Gly Leu Lys Glu Arg Ser Thr Ser Glu Pro Ala Val Pro Pro Glu 50 55 60 Glu Ala Glu Pro His Thr Glu Pro Glu Glu Gln Val Pro Val Glu Ala 65 70 75 80 Glu Pro Gln Asn Ile Glu Asp Glu Ala Lys Glu Gln Ile Gln Ser Leu 85 90 95 Leu His Glu Met Val His Ala Glu His Val Glu Gly Glu Asp Leu Gln 100 105 110 Gln Glu Asp Gly Pro Thr Gly Glu Pro Gln Gln Glu Asp Asp Glu Phe 115 120 125 Leu Met Ala Thr Asp Val Asp Asp Arg Phe Glu Thr Leu Glu Pro Glu 130 135 140 Val Ser His Glu Glu Thr Glu His Ser Tyr His Val Glu Glu Thr Val 145 150 155 160 Ser Gln Asp Cys Asn Gln Asp Met Glu Glu Met Met Ser Glu Gln Glu 165 170 175 Asn Pro Asp Ser Ser Glu Pro Val Val Glu Asp Glu Arg Leu His His 180 185 190 Asp Thr Asp Asp Val Thr Tyr Gln Val Tyr Glu Glu Gln Ala Val Tyr 195 200 205 Glu Pro Leu Glu Asn Glu Gly Ile Glu Ile Thr Glu Val Thr Ala Pro 210 215 220 Pro Glu Asp Asn Pro Val Glu Asp Ser Gln Val Ile Val Glu Glu Val 225 230 235 240 Ser Ile Phe Pro Val Glu Glu Gln Gln Glu Val Pro Pro Asp Thr 245 250 255 <210> 2 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 Glu Pro Ala 1 <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Val Pro Pro Glu 1 <210> 4 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Glu Pro His 1 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 Glu Pro Glu 1 <210> 6 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Val Pro Val 1 <210> 7 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 Glu Pro Gln 1 <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 Gly Pro Thr 1 <210> 9 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Asn Pro Asp 1 <210> 10 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Glu Pro Val 1 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Glu Pro Leu 1 <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Ala Pro Pro Glu 1 <210> 13 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 13 Asn Pro Val 1 <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 14 Phe Pro Val 1 <210> 15 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 15 Val Pro Pro Asp 1 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 16 Glu Pro Ala Val Pro Pro Glu 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 Val Pro Pro Glu Glu Ala Glu Pro His 1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 18 Glu Pro His Thr Glu Pro Glu 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 Glu Pro Glu Glu Gln Val Pro Val 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Val Pro Val Glu Ala Glu Pro Gln 1 5 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 21 Gly Pro Thr Gly Glu Pro Gln 1 5 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 22 Asn Pro Asp Ser Ser Glu Pro Val 1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 23 Ala Pro Pro Glu Asp Asn Pro Val 1 5 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 24 Phe Pro Val Glu Glu Gln Gln Glu Val Pro Pro Asp 1 5 10 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 25 Asp Gly Pro Thr Gly Glu Pro Gln Gln Glu 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 26 Glu Gln Glu Asn Pro Asp Ser Ser Glu Pro Val 1 5 10 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 27 Ala Pro Pro Glu Asp Asn Pro Val Glu Asp 1 5 10 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 28 Glu Glu Gln Gln Glu Val Pro Pro Asp 1 5 <210> 29 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 29 Gly Glu Asp Leu Gln Gln Glu Asp Gly Pro Thr Gly Glu Pro Gln Gln 1 5 10 15 Glu Asp Asp Glu Phe Leu 20 <210> 30 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 30 Asp Met Glu Glu Met Met Ser Glu Gln Glu Asn Pro Asp Ser Ser Glu 1 5 10 15 Pro Val Val Glu Asp Glu 20 <210> 31 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 31 Asn Glu Gly Ile Glu Ile Thr Glu Val Thr Ala Pro Pro Glu Asp Asn 1 5 10 15 Pro Val Glu Asp Ser Gln 20 <210> 32 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 32 Asp Ser Gln Val Ile Val Glu Glu Val Ser Ile Phe Pro Val Glu Glu 1 5 10 15 Gln Gln Glu Val Pro Pro Asp 20 <210> 33 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 33 Lys Ser Ser Gln Ser Leu Val Tyr Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 34 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 35 Leu Gln Gly Ser His Phe Pro Arg Thr 1 5 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 36 Gly Phe Thr Phe Ser Arg Tyr Thr Met Ser 1 5 10 <210> 37 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 37 Tyr Ile Ser Asn Gly Gly Arg Ser Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 38 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 38 Ala Met Asp Asn 1

Claims

1. An isolated anti-Labyrinthin construct comprising an antibody portion that specifically binds to Labyrinthin or a portion thereof and an effector domain.

2. 2. The isolated anti-Labyrinthin construct of claim 1, wherein the antibody moiety is conjugated to an effector domain.

3. The isolated anti-Labyrinthin construct of claim 2 , wherein the effector domain comprises an effector molecule.

4. The isolated anti-Labyrinthin construct of claim 3 , wherein the effector molecule comprises a therapeutic agent.

5. 5. The isolated anti-Labyrinthin construct of claim 4, wherein the therapeutic agent is selected from the group consisting of drugs, toxins, radioisotopes, proteins, peptides, and nucleic acids.

6. 6. The isolated anti-Labyrinthin construct of claim 1 , wherein the isolated anti-Labyrinthin construct is an antibody drug conjugate (ADC).

7. The isolated anti-Labyrinthin construct of claim 3 , wherein the effector domain comprises a diagnostic agent.

8. 8. The isolated anti-Labyrinthin construct of claim 7, wherein the diagnostic agent is a label.

9. 2. The isolated anti-Labyrinthin construct of claim 1, wherein the antibody portion is fused to an effector domain.

10. The isolated anti-Labyrinthin construct of claim 9, wherein the isolated anti-Labyrinthin construct is a chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antibody portion fused to an effector domain, wherein the effector domain comprises a transmembrane domain and an intracellular signaling domain.

11. The isolated anti-Labyrinthin construct of claim 9, wherein the isolated anti-Labyrinthin construct is a bispecific T cell engager (BiTE) comprising an extracellular domain comprising an antibody portion fused to an anti-CD3 antibody or a fragment thereof.

12. 10. The isolated anti-Labyrinthin construct of any one of claims 1 to 9, wherein the isolated anti-Labyrinthin construct is a T cell receptor (TCR) comprising an extracellular domain comprising an antibody portion fused to an effector domain, wherein the effector domain comprises a transmembrane domain and / or an intracellular signaling domain of a TCR subunit.

13. 13. The isolated anti-Labyrinthin construct of any one of claims 1 to 12, wherein the antibody portion is a full-length antibody, a Fab, a Fab', a (Fab')2, an Fv, or a single-chain Fv (scFv).

14. 14. The isolated anti-Labyrinthin construct of any one of claims 1 to 13, wherein the antibody portion binds to Labyrinthin or a portion thereof with a Kd of about 0.1 pM to about 500 nM.

15. 15. The isolated anti-Labyrinthin construct of any one of claims 1 to 14, wherein the antibody portion specifically binds to a Labyrinthin-derived peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 32.

16. 16. The isolated anti-Labyrinthin construct of claim 15, wherein the Labyrinthin-derived peptide comprises a B-cell epitope.

17. 17. The isolated anti-Labyrinthin construct of claim 15 or 16, wherein the Labyrinthin-derived peptide comprises a T-cell epitope.

18. 18. The isolated anti-Labyrinthin construct of any one of claims 1 to 17, wherein the antibody portion is multispecific.

19. The isolated anti-Labyrinthin construct of claim 18, wherein the multispecific antibody portion is a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock-and-lock (DNL) antibody, a chemically cross-linked antibody, a heteromultimeric antibody, or a heteroconjugate antibody.

20. 20. The isolated anti-Labyrinthin construct of claim 19, wherein the multispecific antibody portion is a tandem scFv comprising two scFvs linked by an optional peptide linker.

21. 21. A pharmaceutical composition comprising an isolated anti-Labyrinthin construct according to any one of claims 1 to 20.

22. A host cell expressing an isolated anti-Labyrinthin construct according to any one of claims 1 to 20.

23. 21. A nucleic acid encoding a polypeptide component of the isolated anti-Labyrinthin construct of any one of claims 1 to 20.

24. An effector cell comprising the nucleic acid of claim 23.

25. 25. The effector cell of claim 24, wherein the effector cell is a T cell.

26. A method for detecting a cell presenting labyrinthin or a part thereof on its surface, comprising the steps of: (a) contacting a cell with an isolated anti-Labyrinthin construct according to claim 8; and (b) detecting the presence of the label on the cells A method comprising:

27. 1. A method of treating an individual having a Labyrinthin-positive disease, comprising: (a) an effective amount of the pharmaceutical composition of claim 21; or (b) an effective amount of the effector cell of claim 24 or 25. to an individual.

28. 28. The method of claim 27, wherein the labyrinthine status is used as a criterion for selecting individuals for treatment.

29. 1. A method for diagnosing an individual having a Labyrinthin-positive disease, comprising: (a) administering to an individual an effective amount of an isolated anti-Labyrinthin construct according to claim 8; and (b) determining the level of the label in the individual, where a level of the label above a threshold level indicates that the individual has Labyrinthin-positive disease. A method comprising:

30. 1. A method for diagnosing an individual having a Labyrinthin-positive disease, comprising: (a) contacting a sample from an individual with an isolated anti-Labyrinthin construct according to any one of claims 1 to 20; and (b) identifying one or more cells in the sample that bind the isolated anti-Labyrinthin construct; thereby diagnosing an individual having a Labyrinthin-positive disease.

31. 30. The method according to any one of claims 25 to 29, wherein the labyrinthin-positive disease is a labyrinthin-positive cancer.

32. The method of claim 31 , wherein the labyrinthin-positive cancer is an adenocarcinoma.

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