Anti adam12 antibody and chimeric antigen receptor, and composition and method including the same

Abstract

To provide an anti ADAM12 antibody.SOLUTION: Provided are anti ADAM12 agents such as an anti ADAM12 antibody (Ab), an antigen-binding Ab fragment, a multi-specific Ab and an antigen-binding Ab fragment, an antibody-drug conjugate (ADC), and chimeric antigen receptor (CAR). Further, provided are a nucleic acid sequence and a vector encoding such an anti ADAM12 agent, a cell and a pharmaceutical composition containing such an anti ADAM12 agent, and a method for expanding such a cell. Also provided are a method for treating, preventing or diagnosing a disease such as cancer, and a method for stimulating immune response by using such a material.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 821,257, filed March 20, 2019, and entitled "ANTI-ADAM12 ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS, AND COMPOSITIONS AND METHODS COMPRISING," the entire contents of which are incorporated herein by reference in their entirety.

[0002] Disclosure of sequence listing This application includes as part of its disclosure a biological sequence listing concurrently submitted via EFS-Web. The biological sequence listing described above is contained in a file entitled "1156867o001613.txt", which was created on February 25, 2020, is 204,342 bytes in size, and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to anti-ADAM12 agents, such as anti-ADAM12 antibodies (Abs), antigen-binding Ab fragments, multispecific Abs and antigen-binding Ab fragments, antibody-drug conjugates (ADCs), and chimeric antigen receptors (CARs). The present disclosure also relates to nucleic acid sequences and vectors encoding such anti-ADAM12 agents, cells and pharmaceutical compositions comprising such anti-ADAM12 agents, and methods for expanding such cells. The present disclosure further relates to methods of treating subjects using such anti-ADAM12 agents and compositions, as well as methods of treating, preventing, or diagnosing diseases such as cancer, and methods of stimulating immune responses. The present invention also relates to methods of producing such anti-ADAM12 agents or compositions. [Background technology]

[0004] Immunotherapy is a growing field that allows for the treatment of a variety of diseases for which there were no effective treatment options before. Many examples of immunotherapy have been used in oncology, including antibody therapies targeting CD20 (non-Hodgkin's lymphoma), HER2 (HER2-positive breast cancer), and immune checkpoints such as PD-1, PD-L1, and CTLA-4 (various cancers). Various immunotherapies are also being developed, tested, and / or marketed for non-cancer disease indications, such as autoimmune diseases (Wraith DCet al,Front Immunol.2017 Nov 28;8:1668. doi:10.3389 / fimmu.2017.01668.eCollection 2017).

[0005] Chimeric antigen receptor (CAR) T-cell therapy is a new and emerging type of immunotherapy in which a patient's lymphocytes are genetically modified to express receptors that allow the recognition of specific antigens. Upon antigen recognition, these modified T cells are activated via signaling domains, transforming them into potent cell killers. In 2017, an anti-CD19 CAR T-cell product received FDA approval for B-cell lymphoma, indicating the potential of this therapeutic approach in cancer (e.g., hematological cancers) (Leyfman Y,et la.,Cancer Cell Int.2018 Nov 14;18:182.doi:10.1186 / s12935-018-0685-x.eCollection 2018). While showing great promise for the treatment of solid tumors (Louis CU et al., Blood. 2011 Dec 1;118(23):6050-6056. Prepublished online October 7, 2011, doi:10.1182 / blood-2011-05-354449), CAR T cell therapy faces additional challenges in these indications, such as the presence of an immunosuppressive tumor microenvironment, challenges in accessing the tumor, and the lack of tumor-selective targeting required to minimize "off-tumor" toxicity (Yong CSM et al., Immunol Cell Biol. 2017 Apr;95(4):356-363. doi:10.1038 / icb.2016.128. Epub 2016 Dec 22).

[0006] The ADAM family of enzymes (a disintegrin and metalloproteinase family) are multifunctional, generally membrane-bound zinc proteases (Nyren-Erickson EKBiochim Biophys Acta. 2013 Oct;1830(10):4445-55. doi:10.1016 / j.bbagen.2013.05.011. Epub 2013 May 13). ADAM12 (also known as ADAM metallopeptidase domain 12, a disintegrin and metalloproteinase-12, a disintegrin and metalloproteinase domain-containing protein 12, ADAM12-OT1, CAR10, MCMP, MCMPMltna, Meltrin-a, MLTN, or MLTNA) is a member of the ADAM family. In humans, ADAM12 is encoded by the ADAM12 gene on chromosome 10, with gene location 10q26.2 (NCBI), and there are two naturally occurring ADAM12 splice variants designated ADAM12-L and ADAM12-S (Kveiborg M. et al., Int J Biochem Cell Biol. 2008; 40(9): 1685-702. doi: 10.1016 / j.biocel.2008.01.025. Epub 2008 Feb 1). The ADAM12-L domain composition resembles the prototypical transmembrane ADAM proteins, containing extracellular pro-, metalloproteinase, disintegrin-like, cysteine-rich, and epidermal growth factor (EGF)-like domains followed by a transmembrane domain and a cytoplasmic tail domain. ADAM12-S (soluble splice variant) contains the same domains as ADAM12-L, except that the transmembrane and cytoplasmic domains are replaced by a unique extension of 33 amino acids in the C-terminus.

[0007] Although ADAM12 expression in healthy tissues is low, the general biological roles of ADAM12 are in cell adhesion and fusion, extracellular matrix remodeling, and cell signaling (Nyren-Erickson EKBiochim Biophys Acta. 2013 Oct;1830(10):4445-55. doi:10.1016 / j.bbagen.2013.05.011. Epub 2013 May 13). ADAM12 has been implicated in the pathogenesis of a variety of diseases, including many different types of cancer. Summary of the Invention

[0008] The present invention relates to anti-ADAM12 agents.

[0009] In some embodiments, the anti-ADAM12 agent is an antibody (Ab) or an antigen-binding Ab fragment. The Ab or antigen-binding Ab fragment may bind to ADAM12 and comprises (a) a heavy chain (HC) variable domain (VH) and (b) a light chain (LC) variable domain (VL). The VH may comprise a HC complementarity determining region (CDR) 1 (also referred to as CDR-H1), a HC CDR 2 (also referred to as CDR-H2), a HC CDR 3 (also referred to as CDR-H3), and a human-like HC framework region. The VL may comprise a LC CDR 1 (CDR-L1), a LC CDR 2 (CDR-L2), a LC CDR 3 (CDR-L3), and a human-like LC framework region.

[0010] In some embodiments, HC CDR 1, HC CDR 2, and HC CDR 3 may comprise the amino acid sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively, and LC CDR 1, LC CDR 2, and LC CDR 3 may comprise the amino acid sequences set forth in SEQ ID NOs: 136, 137, and 138, respectively. HC CDR 1, HC CDR 2, and HC CDR 3 may comprise the amino acid sequences encoded by SEQ ID NOs: 232, 233, and 234, respectively, and LC CDR 1, LC CDR 2, and LC CDR 3 may comprise the amino acid sequences encoded by SEQ ID NOs: 236, 237, and 238, respectively. Alternatively, HC CDR 1, HC CDR 2, and HC CDR 3 may comprise the amino acid sequences set forth in SEQ ID NOs: 142, 143, and 144, respectively, and LC CDR 1, LC CDR 2, and LC CDR 3 may comprise the amino acid sequences set forth in SEQ ID NOs: 146, 147, and 148, respectively. HC CDR 1, HC CDR 2, and HC CDR 3 may comprise the amino acid sequences encoded by SEQ ID NOs: 242, 243, and 244, respectively, and LC CDR 1, LC CDR 2, and LC CDR 3 may comprise the amino acid sequences encoded by SEQ ID NOs: 246, 247, and 248, respectively.

[0011] In some embodiments, the human-like HC framework may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human HC framework. The human-like LC framework may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human LC framework.

[0012] In certain embodiments, the HC variable domain may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:131, and the LC variable domain may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:135. The HC variable domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:231, and the LC variable domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:235. Alternatively, the HC variable domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:141, and the LC variable domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:145.The HC variable domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:241, and the LC variable domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:245.

[0013] In some embodiments, the Ab or antigen-binding Ab fragment can be, for example, but not limited to, a monoclonal Ab, a monospecific Ab, a bispecific Ab, a multispecific Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain-specific Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, a CDR-grafted Ab, a fragment antigen-binding (Fab), a F(ab')2, a Fab' fragment, a variable fragment (Fv), a single chain Fv (scFv) fragment, an Fd fragment, a diabody, or a minibody.

[0014] In certain embodiments, the antibody or antigen-binding Ab fragment may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 139, 140, 149, or 150, or (ii) the amino acid sequence encoded by SEQ ID NO: 239, 240, 249, or 250.

[0015] In some embodiments, Ab or antigen-binding Ab fragment may comprise two or more binding specificities. The first specificity may be specific to an epitope of ADAM12. In one embodiment, the second specificity may be specific to another epitope of ADAM12. In another embodiment, the second specificity may be specific to an epitope of a second antigen other than ADAM12. In certain embodiments, the second antigen may be, for example, but not limited to, CD3, NKG2D, or 4-1BB.

[0016] In some embodiments, the Ab or antigen-binding Ab fragment may comprise a human-like fragment crystallizable (Fc) region. The human-like Fc region may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region. The human-like Fc region may bind to an Fc receptor (FcR). The FcR may be, for example, but not limited to, an Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIB1, FcgRIIB2, FcgRIIIA, FcgRIIIB, an Fc epsilon receptor (FceR), FceRI, FceRII, an Fc alpha receptor (FcaR), FcaRI, an Fc alpha / mu receptor (Fca / mR), and a neonatal Fc receptor (FcRn).

[0017] In some embodiments, the anti-ADAM12 agents of the invention are antibody-drug conjugates (ADCs). The ADCs can include (a) any of the Abs or antigen-binding Ab fragments described above, and (b) a drug conjugated to the Ab or antigen-binding Ab fragment.

[0018] In some embodiments, the drug may be, for example, but not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an antibacterial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an antifibrotic drug, an immunosuppressant drug, a steroid, a bronchodilator, a beta-blocker, a matrix metalloproteinase inhibitor, an ADAM12 inhibitor, an ADAM12 signaling inhibitor, an enzyme, a hormone, a neurotransmitter, a toxin, a radioisotope, a chemical compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, a shRNA, a microRNA, an oligonucleotide, or an imaging drug.

[0019] In some embodiments, the ADC is selected from the group consisting of doxorubicin, daunorubicin, cucurbitacin, chaetocin, chaetoglobocin, chlamydocin, calicheamicin, nemorubicin, cryptophycin, menthacarcin, ansamitocin, mitomycin C, geldanamycin, mekelcarmycin, rebeccamycin, safracin, okilactomycin, oligomycin, actinomycin, sandramycin, hypothemycin, polyketomycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, taltobuline, lactacystin, dolastatin, auristatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), telomestatin, tubastatin A, combretastatin, maytansinoid, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodiazepines, SN-38, Ro 5-3335, pwainaphycin, duocarmycin, bafilomycin, taxoid, tubulysin, ferulenol, luciole A, fumagillin, hygrolysin, glucopyericidin, amanitin, ansatrienin, sinerubin, phallacidin, phalloidin, phytosphingosine, piericidin, poronetin, fodophyllotoxin, gramicidin A, sanguinarine, sinefungin, elboxidiene, microcorrin B, microcystin, muscotoxin A, torytoxin, torypolin A, The agent may include a drug selected from the group consisting of myoseverin, mitoxin B, nocuolin A, pseudodoralic acid B, pseurotin A, cyclopamine, curbulin, colchicine, aphidicolin, englerin, cordycepin, apoptolidin, epothilone A, limaquinone, isatropolone, isofistularin, quinaldopeptin, ixabepilone, aeroprisinin, arruginosin, agrochelin, epothilone, and derivatives of any one of the above.

[0020] In some embodiments, the anti-ADAM12 agent of the present invention is a chimeric antigen receptor (CAR). CAR can include (a) an AB domain that binds to ADAM12, (b) a transmembrane (TM) domain, (c) an intracellular signaling (ICS) domain, (d) optionally a hinge that connects the AB domain and the TM domain, and (e) optionally one or more costimulatory (CS) domains.

[0021] In some embodiments, the AB domain can be any Ab or antigen-binding Ab fragment described above.

[0022] In one embodiment, the AB domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 139, 140, 149, or 150, or (ii) the amino acid sequence encoded by SEQ ID NO: 239, 240, 249, or 250.

[0023] In yet another embodiment, the AB domain can compete for binding to ADAM12 with an scFv comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 139, 140, 149, or 150, or (ii) the amino acid sequence encoded by SEQ ID NO: 239, 240, 249, or 250.

[0024] In certain embodiments, the AB domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ADAM12-binding domain of a naturally occurring ADAM12-binding molecule. In one embodiment, the naturally occurring ADAM12-binding molecule may be, for example, but not limited to, alpha actinin 2 (ACTN2), insulin-like growth factor binding protein 3 (IGFBP3), IGFBP-5, phosphatidylinositol 3 kinase regulatory subunit alpha (PIK3R1), heparin-binding epidermal growth factor (HB-EGF), epidermal growth factor (EGF), betacellulin, delta-like 1, placental leucine aminopeptidase (P-LAP), and matrix metalloprotease 14 (MMP-14).

[0025] In some embodiments, the TM domain can be derived from, for example, but not limited to, the TM region of CD28, CD3e, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCRa, TCRb, and CD3z, or a transmembrane portion thereof.

[0026] In certain embodiments, the TM domain may be derived from the TM region of CD28, or a transmembrane portion thereof. The TM domain may optionally comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 161, or (ii) the amino acid sequence encoded by SEQ ID NO: 261.

[0027] In some embodiments, the ICS domain can be derived from, for example, but not limited to, the cytoplasmic signaling sequence of CD3z, lymphocyte receptor chain, TCR / CD3 complex protein, Fc receptor (FcR) subunit, IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, or DAP12, or a functional fragment thereof.

[0028] In certain embodiments, the ICS domain may be derived from the cytoplasmic signaling sequence of CD3z, or a functional fragment thereof. The ICS domain may optionally comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 162, or (ii) the amino acid sequence encoded by SEQ ID NO: 262.

[0029] In some embodiments, the hinge may be derived from CD28. The hinge may optionally comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 163, or (ii) the amino acid sequence encoded by SEQ ID NO: 263.

[0030] In some embodiments, at least one of the one or more CS domains is selected from the group consisting of, for example, but not limited to, CD28, DAP10, 4-1BB (CD137), CD2, CD4, CD5, CD7, CD8a, CD8b, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD69, CD 84, CD96 (Tactile), CD100 (SEMA4D), CD103, OX40 (CD134), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55) , SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B7-H3, BAFFR, BTLA , BLAME (SLAMF8), CEACAM1, CDS, CRTAM, GADS, GITR, HVEM (LIGHTER), IA4, ICAM-1, IL2Rb, IL2Rg, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, ​​NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, or the cytoplasmic signaling sequence of CD83 ligand or a functional fragment thereof.

[0031] In certain embodiments, the CS domain may be derived from the cytoplasmic signaling sequence of CD28, 4-1BB, or DAP10, or a functional fragment thereof. The CS domain may optionally comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 164, or (ii) the amino acid sequence encoded by SEQ ID NO: 264, (iii) the amino acid sequence set forth in SEQ ID NO: 165, (iv) the amino acid sequence encoded by SEQ ID NO: 265, (v) the amino acid sequence set forth in SEQ ID NO: 166, or (vi) the amino acid sequence encoded by SEQ ID NO: 266.

[0032] In certain embodiments, the CAR comprises the amino acid sequence of: (i) h6E6scFvHL-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 171); (ii) h6E6scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 172); (iii) h6E6scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 173); (iv) h6E6scFvLH-CD28H-CD (v) the amino acid sequence of h6E6scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 175); (vi) the amino acid sequence of h6E6scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 176); (vii) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 177); (viii) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 178), (ix) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 179), (x) the amino acid sequence of h6C10scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 180), (xi) h6C10scFvLH-CD28H-CD28TM-41BB (xii) the amino acid sequence of h6C10scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 182); or (xiii) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 271-282.

[0033] In some embodiments, the CAR may further comprise a cytotoxic drug conjugated to the AB domain.

[0034] The present invention relates to an isolated nucleic acid sequence encoding any of the anti-ADAM12 agents described above.

[0035] In some embodiments, the isolated nucleic acid sequence may encode an antibody (Ab) or an antigen-binding Ab fragment. The Ab or antigen-binding Ab fragment may bind to ADAM12 and include an HC variable domain (VH) and an LC variable domain (VL). The VH may include an HC complementarity determining region (CDR) 1, an HC CDR 2, an HC CDR 3, and a human-like HC framework. The VL may include an LC CDR 1, an LC CDR 2, an LC CDR 3, and a human-like LC framework region.

[0036] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are HC CDR 1, HC CDR 2, and LC CDR 3 may comprise the amino acid sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively, and LC CDR 1, LC CDR 2, and LC CDR 3 may comprise the amino acid sequences set forth in SEQ ID NOs: 136, 137, and 138, respectively. HC CDR 1, HC CDR 2, and HC CDR 3 may comprise the amino acid sequences encoded by SEQ ID NOs: 232, 233, and 234, respectively, and LC CDR 1, LC CDR 2, and LC CDR 3 may comprise the amino acid sequences encoded by SEQ ID NOs: 236, 237, and 238, respectively. Alternatively, HC CDR 1, HC CDR 2, and HC CDR 3 may comprise the amino acid sequences set forth in SEQ ID NOs: 142, 143, and 144, respectively, and LC CDR 1, LC CDR 2, and LC CDR 3 may comprise the amino acid sequences set forth in SEQ ID NOs: 146, 147, and 148, respectively. HC CDR 1, HC CDR 2, and HC CDR 3 may comprise the amino acid sequences encoded by SEQ ID NOs: 242, 243, and 244, respectively, and LC CDR 1, LC CDR 2, and LC CDR 3 may comprise the amino acid sequences encoded by SEQ ID NOs: 246, 247, and 248, respectively.

[0037] In some embodiments, the human-like HC framework may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human HC framework. The human-like LC framework may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human LC framework.

[0038] In certain aspects, the isolated nucleic acid sequence may encode an Ab or antigen-binding Ab fragment, wherein the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:131 and the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:135. The VH may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:231, and the VL may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:235. Alternatively, the VH may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:141, and the VL may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:145.The VH may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:241, and the VL may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:245.

[0039] In some embodiments, the isolated nucleic acid sequence may encode an Ab or antigen-binding Ab fragment that is, for example, but not limited to, a monoclonal Ab, a monospecific Ab, a bispecific Ab, a multispecific Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain-specific Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, a CDR-grafted Ab, a fragment antigen-binding (Fab), a F(ab')2, a Fab' fragment, a variable fragment (Fv), a single chain Fv (scFv) fragment, an Fd fragment, a diabody, or a minibody.

[0040] In certain embodiments, the isolated nucleic acid sequence may encode an Ab or antigen-binding Ab fragment that contains amino acids at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 139, 140, 149, or 150, or (ii) the amino acid sequence encoded by SEQ ID NO: 239, 240, 249, or 250.

[0041] In some embodiments, the isolated nucleic acid sequence may encode an Ab or antigen-binding Ab fragment that comprises two or more binding specificities. The first specificity may be specific to an epitope of ADAM12. In one embodiment, the second specificity may be specific to another epitope of ADAM12. In another embodiment, the second specificity may be specific to an epitope of a second antigen other than ADAM12. In certain embodiments, the second antigen may be, for example, but not limited to, CD3, NKG2D, or 4-1BB.

[0042] In some embodiments, the isolated nucleic acid can encode an Ab or antigen-binding Ab fragment that includes a human-like fragment crystallizable (Fc) region. In certain embodiments, the human-like Fc region can be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region.

[0043] The human-like Fc region can bind to an Fc receptor (FcR). The FcR can be, for example, but not limited to, an Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIB1, FcgRIIB2, FcgRIIIA, FcgRIIIB, an Fc epsilon receptor (FceR), FceRI, FceRII, an Fc alpha receptor (FcaR), FcaRI, an Fc alpha / mu receptor (Fca / mR), or a neonatal Fc receptor (FcRn).

[0044] In some embodiments, the isolated nucleic acid sequence may encode any of the CARs described above. The CAR may include (a) an AB domain that binds to ADAM12, (b) a transmembrane (TM) domain, (c) an intracellular signaling (ICS) domain, (d) optionally a hinge that connects the AB domain and the TM domain, and (e) optionally one or more costimulatory (CS) domains.

[0045] In some embodiments, the isolated nucleic acid sequence may encode a CAR in which the AB domain is encoded by any of the nucleic acid sequences encoding the AB domains described above.

[0046] In certain embodiments, the isolated nucleic acid sequence may encode a CAR in which the AB domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 139, 140, 149, or 150, or (ii) the amino acid sequence encoded by SEQ ID NO: 239, 240, 249, or 250.

[0047] In certain embodiments, the isolated nucleic acid sequence may encode a CAR that competes for binding to ADAM12 with an scFv whose AB domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by (i) SEQ ID NO: 139, 140, 149, or 150, or (ii) SEQ ID NO: 239, 240, 249, or 250.

[0048] In certain aspects, the isolated nucleic acid sequence may encode a CAR in which the AB domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ADAM12-binding domain of a naturally occurring ADAM12-binding molecule. In certain embodiments, the naturally occurring ADAM12-binding molecule may be, for example, but not limited to, alpha actinin 2 (ACTN2), insulin-like growth factor binding protein 3 (IGFBP3), IGFBP-5, phosphatidylinositol 3 kinase regulatory subunit alpha (PIK3R1), heparin-binding epidermal growth factor (HB-EGF), epidermal growth factor (EGF), betacellulin, delta-like 1, placental leucine aminopeptidase (P-LAP), and matrix metalloprotease 14 (MMP-14).

[0049] In some embodiments, the isolated nucleic acid sequence may encode a CAR in which the TM domain is derived from, for example, but not limited to, the TM region of CD28, CD3e, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCRa, TCRb, and CD3z, or a transmembrane portion thereof. In certain embodiments, the TM domain may be derived from the TM region of CD28, or a transmembrane portion thereof. The TM domain may optionally comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 161, or (ii) the amino acid sequence encoded by SEQ ID NO: 261.

[0050] In some embodiments, the isolated nucleic acid sequence may encode a CAR in which the ICS domain is derived from the cytoplasmic signaling sequence of, for example, but not limited to, CD3z, lymphocyte receptor chain, TCR / CD3 complex protein, Fc receptor (FcR) subunit, IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, and DAP12, or a functional fragment thereof. In certain embodiments, the ICS domain may be derived from the cytoplasmic signaling sequence of CD3z, or a functional fragment thereof. The ICS domain may optionally comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 162, or (ii) the amino acid sequence encoded by SEQ ID NO: 262.

[0051] In some embodiments, the isolated nucleic acid sequence may encode a CAR in which the hinge is derived from CD28. The hinge may optionally comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 163, or (ii) the amino acid sequence encoded by SEQ ID NO: 263.

[0052] In some embodiments, the isolated nucleic acid sequence comprises at least one of one or more CS domains, such as, but not limited to, CD28, DAP10, 4-1BB (CD137), CD2, CD4, CD5, CD7, CD8a, CD8b, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD19g, CD29h, CD30h, CD40i, CD49j, CD49k, CD49f, CD19i, CD29j, CD29k, CD30h, CD40i, CD49j, CD49k ... D69, CD84, CD96(Tactile), CD100(SEMA4D), CD103, OX40(CD134), SLAM(SLAMF1, CD150, IPO-3), CD160(B Y55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B7-H3, BAFFR, BTL A CAR may be encoded by a cytoplasmic signaling sequence of A, BLAME (SLAMF8), CEACAM1, CDS, CRTAM, GADS, GITR, HVEM (LIGHTER), IA4, ICAM-1, IL2Rb, IL2Rg, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, ​​NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, or a functional fragment thereof. In certain embodiments, the CS domain may be derived from the cytoplasmic signaling sequence of CD28, 4-1BB, or DAP10, or a functional fragment thereof.The CS domain may optionally comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to (i) the amino acid sequence set forth in SEQ ID NO: 164, or (ii) the amino acid sequence encoded by SEQ ID NO: 264, (iii) the amino acid sequence set forth in SEQ ID NO: 165, (iv) the amino acid sequence encoded by SEQ ID NO: 265, (v) the amino acid sequence set forth in SEQ ID NO: 166, or (vi) the amino acid sequence encoded by SEQ ID NO: 266.

[0053] In some embodiments, the isolated nucleic acid sequence is selected from the group consisting of: (i) the amino acid sequence of h6E6scFvHL-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 171); (ii) the amino acid sequence of h6E6scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 172); (iii) the amino acid sequence of h6E6scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 173); (iv) the amino acid sequence of h6E6scFvLH-CD28 (v) the amino acid sequence of h6E6scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 175); (vi) the amino acid sequence of h6E6scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 176); (vii) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 177); (viii) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 178), (ix) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 179), (x) the amino acid sequence of h6C10scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 180), (xi) the amino acid sequence of h6C10scFvLH-CD28H-CD28TM-41BBCS-C The CAR may encode a CAR comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of D3zICS (SEQ ID NO: 181), (xii) the amino acid sequence of h6C10scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 182), or (xiii) the amino acid sequence encoded by any one of SEQ ID NOs: 271-282.

[0054] In some embodiments, any of the above isolated nucleic acid sequences may further comprise a leader sequence (LS). In one aspect, the LS is optionally at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:260.

[0055] In some embodiments, any one of the above isolated nucleic acid sequences may further comprise a sequence encoding a T2A sequence and / or a truncated CD19 (trCD19). In one aspect, the T2A sequence is optionally at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:269. In one aspect, the trCD19 may optionally comprise an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:170. In certain aspects, the trCD19 may be encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:270.

[0056] The present invention relates to vectors comprising any nucleic acid sequence encoding an anti-ADAM12 agent.

[0057] In some embodiments, a vector may include any of the nucleic acid sequences described above.

[0058] In some aspects, the vector can be, for example, but not limited to, a DNA, RNA, plasmid, cosmid, viral vector, lentiviral vector, adenoviral vector, or retroviral vector.

[0059] The present invention relates to recombinant or isolated cells.

[0060] In some embodiments, the recombinant or isolated cell may comprise (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (iii) any CAR described above, (iv) any nucleic acid sequence described above, or (v) any vector described above.

[0061] In some aspects, the recombinant or isolated cell can be, for example, but not limited to, a non-mammalian cell, optionally a plant cell, a bacterial cell, a fungal cell, a yeast cell, a protozoan cell, or an insect cell.

[0062] In some aspects, the recombinant or isolated cell can be, for example, but not limited to, a mammalian cell, optionally a human cell, a rat cell, or a mouse cell.

[0063] In some aspects, the recombinant or isolated cells can be, for example, but not limited to, stem cells.

[0064] In some aspects, recombinant or isolated cells can be, for example, but not limited to, primary cells, optionally primary human cells, or cells derived therefrom.

[0065] In some aspects, the recombinant or isolated cell can be, for example, but not limited to, a cell line, optionally a hybridoma cell line.

[0066] In some aspects, the recombinant or isolated cell can be, for example, but not limited to, an immune cell.

[0067] In some embodiments, the recombinant or isolated cells can be MHC+ or MHC-.

[0068] In some embodiments, recombinant or isolated cells include, for example, but not limited to, cell lines, T cells, T cell precursors, CD4+ T cells, helper T cells, regulatory T cells, CD8+ T cells, naive T cells, effector T cells, memory T cells, stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, cytotoxic T cells, mucosal associated invariant T (MAIT) cells, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, and a / b T cells, g / d T cells, and / or β T cells. The cells may be T cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, perforin-deficient cells, granzyme-deficient cells, B cells, myeloid cells, monocytes, macrophages, or dendritic cells.

[0069] In certain embodiments, the recombinant or isolated cells may be T cells or T cell precursors.

[0070] In certain aspects, the recombinant or isolated cell may be a T cell that has been modified such that its endogenous T cell receptor (TCR) is (i) not expressed, (ii) not functionally expressed, or (iii) expressed at a reduced level compared to a wild-type T cell.

[0071] In certain embodiments, the recombinant or isolated cells can be activated or stimulated to proliferate when the CAR binds to its target molecule.

[0072] In certain embodiments, the recombinant or isolated cell may exhibit cytotoxicity towards cells expressing a target molecule when the CAR binds to the target molecule.

[0073] In certain embodiments, administration of the recombinant or isolated cells can ameliorate a disease, cancer, cardiac condition, autoimmune condition, inflammatory condition, or fibrotic condition when the CAR binds to its target molecule.

[0074] In certain embodiments, the recombinant or isolated cells can increase the expression of cytokines and / or chemokines when the CAR binds to its target molecule. The cytokine can be IFN-g.

[0075] In certain embodiments, the recombinant or isolated cells can decrease the expression of a cytokine and / or a chemokine when the CAR binds to its target. The cytokine can be TGF-b.

[0076] The present invention relates to a collection of recombinant or isolated cells.

[0077] In some embodiments, the collection may comprise at least any one recombinant or isolated cell described above.

[0078] The present invention relates to pharmaceutical compositions.

[0079] In some embodiments, the pharmaceutical composition may comprise (a) (ai) any Ab or antigen-binding Ab fragment described above, (a-ii) any ADC described above, (iii) any CAR described above, (iv) any nucleic acid sequence described above, (v) any vector described above, (vi) any cell described above, or (vii) any collection of cells, and, optionally, (b) a pharmaceutically acceptable excipient or carrier.

[0080] The present invention relates to a method of treating a subject.

[0081] In some embodiments, the method may be a method of treating a subject, which may comprise administering to a subject in need thereof a therapeutically effective amount of (i) any of the Abs or antigen-binding Ab fragments described above, (ii) any of the ADCs described above, (iii) any of the CARs described above, (iv) any of the nucleic acid sequences described above, (v) any of the vectors described above, (vi) any of the cells described above, (vii) any collection of cells described above, or (viii) any of the pharmaceutical compositions described above.

[0082] In some embodiments, the methods may be used to treat, for example, but not limited to, cancer, fibrosis, autoimmunity, cardiovascular conditions, allergic conditions, respiratory diseases, nephropathy, neurological diseases, muscular diseases, liver diseases, metabolic syndrome, infection, or inflammatory disorders. In certain embodiments, the methods may be used to treat cancer, for example, but not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumors, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, or thyroid cancer.

[0083] In some embodiments, the method may be a method of treating a subject with an anti-ADAM12 agent. The method may include: (a) obtaining or obtaining a biological sample from a subject; (b) measuring the expression level of ADAM12 in the biological sample; (c) determining whether the subject is an ADAM12 overexpressor; and (d) if the subject is an ADAM12 overexpressor, administering to the subject a therapeutically effective amount of (di) any of the Abs or antigen-binding Ab fragments described above, (d-ii) any of the ADCs described above, (d-iii) any of the CARs described above, (d-iv) any of the nucleic acid sequences described above, (dv) any of the vectors described above, (d-vi) any of the cells described above, (d-vii) any of the collections of cells described above, or (d-viii) any of the pharmaceutical compositions described above. In certain aspects, an ADAM12 overexpressor is a subject whose ADAM12 expression is at least 1.5 times higher than that of a normal or healthy subject. In certain embodiments, an ADAM12 overexpressor is a subject whose ADAM12 expression is at least 1.75 times higher than that of a normal or healthy subject. In certain embodiments, an ADAM12 overexpressor is a subject whose ADAM12 expression is at least 2 times higher than that of a normal or healthy subject.

[0084] In some embodiments, the subject may be suffering from cancer, such as, but not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, or thyroid cancer.

[0085] The present invention relates to a method of immune stimulation.

[0086] In some embodiments, the method may be a method for stimulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of (i) any of the Abs or antigen-binding Ab fragments described above, (ii) any of the ADCs described above, (iii) any of the CARs described above, (iv) any of the nucleic acid sequences described above, (v) any of the vectors described above, (vi) any of the cells described above, (vii) any collection of cells described above, or (viii) any of the pharmaceutical compositions described above.

[0087] The present invention relates to a method for treating a disease.

[0088] In some embodiments, the method may be a method of treating a disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (i) any of the Ab or antigen-binding Ab fragment described above, (ii) any of the ADC described above, (iii) any of the CAR described above, (iv) any of the nucleic acid sequence described above, (v) any of the vector described above, (vi) any of the cells described above, (vii) any collection of cells described above, or (viii) any of the pharmaceutical compositions described above.

[0089] In some aspects, the disease can be, for example, but not limited to, cancer, fibrosis, autoimmunity, cardiovascular conditions, allergic conditions, respiratory diseases, nephropathy, neurological diseases, muscular diseases, liver diseases, metabolic syndrome, infection, or inflammatory disorders.

[0090] In certain aspects, the disease may be cancer, which may optionally be bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, or thyroid cancer.

[0091] The present invention further relates to a method for expanding a population of cells.

[0092] In some aspects, the method may be a method of expanding a population of cells in a subject.

[0093] In some embodiments, the method may include administering to a subject (i) any of the nucleic acid sequences described above, (ii) any of the vectors described above, (iii) any of the cells described above, (iv) any of the collections of cells described above, or (v) any of the pharmaceutical compositions described above.

[0094] In some embodiments, administration can result in a population of cells that includes at least one desired cell, e.g., a cell that can include any Ab or Ab fragment described above, any ADC described above, and / or any CAR described above. In certain embodiments, the cell can include a nucleic acid encoding such an Ab or Ab fragment, ADC, or CAR.

[0095] In some embodiments, the resulting population of cells may be maintained in the subject for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 2 years, or at least 3 years following administration.

[0096] In some embodiments, the subject may be suffering from cancer.

[0097] In certain embodiments, the disease may be bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, or thyroid cancer.

[0098] In some embodiments, any of the above methods may further include administering a second agent.

[0099] In certain embodiments, the second agent can be, but is not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an antibacterial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an anti-fibrotic drug, an immunosuppressant drug, a steroid, a bronchodilator, a beta-blocker, a matrix metalloproteinase inhibitor, an ADAM12 inhibitor, an ADAM12 signaling inhibitor, an anti-ADAM12 agent of the present invention, an enzyme, a hormone, a neurotransmitter, a toxin, a radioisotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, a shRNA, a microRNA, an oligonucleotide, or an imaging agent.

[0100] The present invention further relates to methods of generating cells containing any of the above-mentioned CARs.

[0101] In some embodiments, the method comprises: (i) introducing into a cell at least one nucleic acid sequence encoding at least one CAR as described in (ia) any one of the above; or (ib) at least one nucleic acid sequence as described in any one of the above; or (ii) transducing the cells with any one of the vectors described above. Optionally, the method may further comprise (iii) isolating the cells based on the expression of the above-mentioned CAR and / or selectable marker as determined via flow cytometry or immunofluorescence assay. [Brief description of the drawings]

[0102] [Figure 1A] Provided is an exemplary schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1A shows a schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1B shows an exemplary schematic diagram of a CAR construct of the present disclosure, the CAR construct comprises an antigen binding (AB) domain, a transmembrane (TM) domain, an intracellular signaling (ICS) domain, and further comprises a hinge linking the AB and TM domains, and one or two costimulatory (CS) domains. Figure 1C shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector comprises a leader sequence (LS) and an exemplary CAR construct as shown in Figure 1B. Figure 1E shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector further comprises an exemplary ribosomal skip sequence (T2A) and an exemplary expression / purification marker, truncated CD19 (trCD19). [Figure 1B] Provided is an exemplary schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1A shows a schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1B shows an exemplary schematic diagram of a CAR construct of the present disclosure, the CAR construct comprises an antigen binding (AB) domain, a transmembrane (TM) domain, an intracellular signaling (ICS) domain, and further comprises a hinge linking the AB and TM domains, and one or two costimulatory (CS) domains. Figure 1C shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector comprises a leader sequence (LS) and an exemplary CAR construct as shown in Figure 1B. Figure 1E shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector further comprises an exemplary ribosomal skip sequence (T2A) and an exemplary expression / purification marker, truncated CD19 (trCD19). [Figure 1C]Provided is an exemplary schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1A shows a schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1B shows an exemplary schematic diagram of a CAR construct of the present disclosure, the CAR construct comprises an antigen binding (AB) domain, a transmembrane (TM) domain, an intracellular signaling (ICS) domain, and further comprises a hinge linking the AB and TM domains, and one or two costimulatory (CS) domains. Figure 1C shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector comprises a leader sequence (LS) and an exemplary CAR construct as shown in Figure 1B. Figure 1E shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector further comprises an exemplary ribosomal skip sequence (T2A) and an exemplary expression / purification marker, truncated CD19 (trCD19). [Figure 1D] Provided is an exemplary schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1A shows a schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1B shows an exemplary schematic diagram of a CAR construct of the present disclosure, the CAR construct comprises an antigen binding (AB) domain, a transmembrane (TM) domain, an intracellular signaling (ICS) domain, and further comprises a hinge linking the AB and TM domains, and one or two costimulatory (CS) domains. Figure 1C shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector comprises a leader sequence (LS) and an exemplary CAR construct as shown in Figure 1B. Figure 1E shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector further comprises an exemplary ribosomal skip sequence (T2A) and an exemplary expression / purification marker, truncated CD19 (trCD19). [Figure 1E]Provided is an exemplary schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1A shows a schematic diagram of a chimeric antigen receptor (CAR) of the present disclosure. Figure 1B shows an exemplary schematic diagram of a CAR construct of the present disclosure, the CAR construct comprises an antigen binding (AB) domain, a transmembrane (TM) domain, an intracellular signaling (ICS) domain, and further comprises a hinge linking the AB and TM domains, and one or two costimulatory (CS) domains. Figure 1C shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector comprises a leader sequence (LS) and an exemplary CAR construct as shown in Figure 1B. Figure 1E shows an exemplary schematic diagram of a vector construct encoding a CAR of the present disclosure, the vector further comprises an exemplary ribosomal skip sequence (T2A) and an exemplary expression / purification marker, truncated CD19 (trCD19). [Diagram 2] A schematic diagram showing various exemplary AB domain constructs of CARs of some embodiments is shown. The first two examples are "h6E6scFvHL" (or "h6E6 scFv HL") and "h6E6scFvLH" (or "h6E6 scFv LH"), which are scFvs derived from "h6E6" and are humanized versions of the murine anti-ADAM12 antibody 6E6. The next two examples are "h6C10scFvHL" (or "h6C10 scFv HL") and "h6C10scFvLH" (or "h6C10 scFv LH"), which are scFvs derived from "h6C10" and are humanized versions of the murine anti-ADAM12 antibody 6C10. In the last two examples, naturally occurring molecules that bind to ADAM12, or ADAM12-binding portions of such molecules, are used in the AB domain. [Figure 3A]3A includes schematic diagrams of various exemplary CAR constructs of some embodiments of the present invention. In the exemplary construct of FIG. 3A, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, CD28CS is used as the CS domain, and CD3zICS is used as the ICS domain. In the exemplary construct of FIG. 3B, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, 41BBCS is used as the CS domain, and CD3zICS is used as the ICS domain. In the exemplary construct of FIG. 3C, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, DAP10CS is used as the CS domain, and CD3zICS is used as the ICS domain. CD28H is the hinge derived from human CD28. CD28TM is the TM domain derived from human CD28. CD28CS is the CS region derived from the cytoplasmic signaling sequence of human CD28. CD3zICS is the ICS domain derived from human CD3 zeta. As shown in Figure 1C, any of the CAR constructs described in this figure or in this application can be used with LS, T2A, and / or trCD19. [Figure 3B]3A includes schematic diagrams of various exemplary CAR constructs of some embodiments of the present invention. In the exemplary construct of FIG. 3A, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, CD28CS is used as the CS domain, and CD3zICS is used as the ICS domain. In the exemplary construct of FIG. 3B, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, 41BBCS is used as the CS domain, and CD3zICS is used as the ICS domain. In the exemplary construct of FIG. 3C, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, DAP10CS is used as the CS domain, and CD3zICS is used as the ICS domain. CD28H is the hinge derived from human CD28. CD28TM is the TM domain derived from human CD28. CD28CS is the CS region derived from the cytoplasmic signaling sequence of human CD28. CD3zICS is the ICS domain derived from human CD3 zeta. As shown in Figure 1C, any of the CAR constructs described in this figure or in this application can be used with LS, T2A, and / or trCD19. [Figure 3C]3A includes schematic diagrams of various exemplary CAR constructs of some embodiments of the present invention. In the exemplary construct of FIG. 3A, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, CD28CS is used as the CS domain, and CD3zICS is used as the ICS domain. In the exemplary construct of FIG. 3B, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, 41BBCS is used as the CS domain, and CD3zICS is used as the ICS domain. In the exemplary construct of FIG. 3C, one of the AB domains shown in FIG. 2 is used as the AB domain, CD28H is used as the hinge, CD28TM is used as the TM domain, DAP10CS is used as the CS domain, and CD3zICS is used as the ICS domain. CD28H is the hinge derived from human CD28. CD28TM is the TM domain derived from human CD28. CD28CS is the CS region derived from the cytoplasmic signaling sequence of human CD28. CD3zICS is the ICS domain derived from human CD3 zeta. As shown in Figure 1C, any of the CAR constructs described in this figure or in this application can be used with LS, T2A, and / or trCD19. [Figure 4] FIG. 1 shows a flow chart illustrating one of many possible methods for producing isolated recombinant CAR-expressing cells that can be used for in vitro or in vivo assays. [Diagram 5] Histograms of ADAM12 staining of cancer cell lines analyzed by flow cytometry are included. MCF7-ADAM12 cells (human breast cancer cell line transfected with an ADAM12 expression vector) (left) were stained with crude ascites samples from mice harboring cells producing anti-human ADAM12 antibody (clone 7B8), and U87-MG cells (glioblastoma cell line) (right) were stained with crude ascites samples from mice harboring cells producing anti-human ADAM12 antibody (clone 8F8) or with mouse anti-human ADAM12 monoclonal antibody (clone 6C10). [Figure 6] Graphs showing the cytotoxicity of anti-ADAM12 CAR-expressing cells of the present invention are included. MCF7-ADAM12 cells were transduced with luciferase-expressing vector JC7 and used as target cells. Human T cells from donor 1 were transduced with vectors encoding anti-ADAM12 CARs (anti-ADAM12 CAR1 or anti-ADAM12 CAR2) or with an empty vector (EV, i.e., CD19 only), and CD19-positive cells were enriched, expanded, and used as effector cells. Cells were co-cultured at various effector (CAR T cells): target (tumor cells) ratios, and cytotoxicity was evaluated using a luciferase plate assay after 24 hours (top) or 48 hours (bottom) of co-culture. Asterisks represent the significance between anti-ADAM12 CAR and EV T cells using Student's T-test (*p<0.05, **p<0.01, ***p<0.001). [Figure 7A] Contains results from the in vivo efficacy study described in Example 6. NSG mice bearing intraperitoneal MCF7-ADAM12-Luc tumors were treated with human T cells expressing trCD19 but not anti-ADAM12 (EV T) or human T cells expressing anti-ADAM12 CAR1 (CAR1 T). Figure 7A is a series of Xenogen-IVIS® images showing the change in tumor burden in each treatment group. Figure 7B is a graph comparing the average tumor burden in the two treatment groups using luminescence signal intensity (radiance (photons / sec)). Error bars: standard error of the mean (SEM). Statistical differences between the groups were analyzed using Student's T-test with Mann-Whitney rankings (**p<0.01, and ***p<0.001). Figure 7C is a graph comparing the average body weight in the two groups. [Figure 7B]Contains results from the in vivo efficacy study described in Example 6. NSG mice bearing intraperitoneal MCF7-ADAM12-Luc tumors were treated with human T cells expressing trCD19 but not anti-ADAM12 (EV T) or human T cells expressing anti-ADAM12 CAR1 (CAR1 T). Figure 7A is a series of Xenogen-IVIS® images showing the change in tumor burden in each treatment group. Figure 7B is a graph comparing the average tumor burden in the two treatment groups using luminescence signal intensity (radiance (photons / sec)). Error bars: standard error of the mean (SEM). Statistical differences between the groups were analyzed using Student's T-test with Mann-Whitney rankings (**p<0.01, and ***p<0.001). Figure 7C is a graph comparing the average body weight in the two groups. [Figure 7C] Contains results from the in vivo efficacy study described in Example 6. NSG mice bearing intraperitoneal MCF7-ADAM12-Luc tumors were treated with human T cells expressing trCD19 but not anti-ADAM12 (EV T) or human T cells expressing anti-ADAM12 CAR1 (CAR1 T). Figure 7A is a series of Xenogen-IVIS® images showing the change in tumor burden in each treatment group. Figure 7B is a graph comparing the average tumor burden in the two treatment groups using luminescence signal intensity (radiance (photons / sec)). Error bars: standard error of the mean (SEM). Statistical differences between the groups were analyzed using Student's T-test with Mann-Whitney rankings (**p<0.01, and ***p<0.001). Figure 7C is a graph comparing the average body weight in the two groups. [Figure 8] The results from the cytokine production test described in Example 5 are included. Anti-ADAM12 CAR1, anti-ADAM12 CAR2, or EV T cells were cultured with MCF7-ADAM12 for 24 hours, and the concentration of IFN-g in the supernatant was compared. Error bars: standard error of the mean (SEM). Statistical differences in IFN-g levels were calculated using Student's T-test (****p<0.0001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] One aspect of the present invention relates generally to the construction and use of novel ADAM12-binding agents.

[0104] In one embodiment, the anti-ADAM12 agent is, for example, but not limited to, an anti-ADAM12 antibody (Ab), an antigen-binding Ab fragment, a multispecific Ab, a multispecific antigen-binding Ab fragment, an antibody-drug conjugate (ADC), and a chimeric antigen receptor (CAR).

[0105] In one embodiment, the antigen-binding Ab fragment comprises an AB domain.

[0106] In one embodiment, the antigen-binding Ab fragment can be an AB domain.

[0107] The invention also provides polynucleotides encoding such Abs, antigen-binding Ab fragments, multispecific Abs, multispecific antigen-binding Ab fragments, ADCs, or CARs that bind to ADAM12, vectors comprising such polynucleotides, as well as cells comprising such Abs, antigen-binding Ab fragments, multispecific Abs, multispecific antigen-binding Ab fragments, ADCs, or CARs (e.g., polynucleotides, or such vectors).The invention also provides compositions comprising such Abs, antigen-binding Ab fragments, multispecific Abs, multispecific antigen-binding Ab fragments, ADCs, CARs, such polynucleotides, such vectors, or such cells.

[0108] The present invention further provides methods for producing and using Abs, antigen-binding Ab fragments, multispecific Abs, multispecific antigen-binding Ab fragments, ADCs, or CARs that bind to ADAM12, or cells that express Abs, antigen-binding Ab fragments, multispecific Abs, multispecific antigen-binding Ab fragments, ADCs, or CARs that bind to ADAM12.The present invention also provides methods for treating conditions associated with ADAM12 expression in subjects, such as cancer.Such anti-ADAM12 Abs, antigen-binding Ab fragments, multispecific Abs, multispecific antigen-binding Ab fragments, ADCs, CARs, and cells that contain nucleic acid sequences that code such Abs, antigen-binding Ab fragments, multispecific Abs, multispecific antigen-binding Ab fragments, ADCs, or CARs that bind to ADAM12 can be used to treat diseases, disorders, or conditions associated with the unwanted proliferation of cells that express ADAM12.

[0109] binding target In one embodiment, an anti-ADAM12 agent of the invention binds to ADAM12.

[0110] In one embodiment, the target or binding target of an anti-ADAM12 agent of the invention is ADAM12.

[0111] In one aspect, the anti-ADAM12 antibodies (Abs), anti-ADAM12 antigen-binding Ab fragments, anti-ADAM12 multispecific Abs, anti-ADAM12 multispecific antigen-binding Ab fragments, anti-ADAM12 antibody-drug conjugates (ADCs), and anti-ADAM12 chimeric antigen receptors (CARs) of the invention individually comprise an AB domain that binds to ADAM12 (a disintegrin and metalloproteinase-12), also known as ADAM metalloproteinase domain 12, a disintegrin, a disintegrin and metalloproteinase domain-containing protein 12, ADAM12-OT1, CAR10, MCMP, MCMPMltna, Meltrin-a, MLTN, or MLTNA.

[0112] In humans, ADAM12 is encoded by the ADAM12 gene on chromosome 10, with gene location 10q26.2 (NCBI), and there are two naturally occurring ADAM12 splice variants designated ADAM12-L (L for long) and ADAM12-S (S for short) (Kveiborg M. et al., Int J Biochem Cell Biol. 2008;40(9):1685-702. doi:10.1016 / j.biocel.2008.01.025. Epub 2008 Feb 1).

[0113] The ADAM12-L domain composition resembles the prototypical transmembrane ADAM proteins, containing extracellular pro-, metalloproteinase, disintegrin-like, cysteine-rich, and epidermal growth factor (EGF)-like domains followed by a transmembrane domain and a cytoplasmic tail domain. ADAM12-S (a soluble splice variant) contains the same domains as ADAM12-L, except that the transmembrane and cytoplasmic domains are replaced by a unique extension of 33 amino acids in the C-terminus.

[0114] Human ADAM12-L has the amino acid sequence provided as GenBank Accession: AAC08702.2, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, human ADAM12-L has the amino acid sequence provided as SEQ ID NO: 101, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). Human ADAM12-S has the amino acid sequence provided as GenBank Accession: AAC08703.2, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, human ADAM12-S has the amino acid sequence provided as SEQ ID NO: 102, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

[0115] In some embodiments, an anti-ADAM12 agent of the invention binds to ADAM12-L.

[0116] In some embodiments, an anti-ADAM12 agent of the invention binds to ADAM12-S.

[0117] In some embodiments, an anti-ADAM12 agent of the invention binds to both ADAM12-L and ADAM12-S.

[0118] ADAM12 is upregulated and / or plays a pathological role in a wide variety of cancers, including, but not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumors, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, and thyroid cancer (Kveiborg M.et al.,Int J Biochem Cell Biol.2008;40(9):1685-702.doi:10.1016 / j.biocel.2008.01.025.Epub 2008 Feb 1; Le Pabic H.et l.,Hepatology.2003 May;37(5):1056-66, Skubitz KMet al.,J Lab Clin Med.2004 Feb;143(2):89-98., Carl-McGrath S.et al.,Int J Oncol.2005 Jan;26(1):17-24, Mochizuki S.et al.,Cancer Sci.2007 May;98(5):621-8.Epub 2007 Mar 9, Colombo C.et al.,J Pathol.2011 Dec;225(4):574-82.doi:10.1002 / path.2951.Epub 2011 Aug 8, Sookprasert A.et al.,Asian Pac J Cancer Prev.2012;13 Suppl:3-6, Uehara E. et al., Int J Oncol.2012 May;40(5):1414-22.doi:10.3892 / ijo.2012.1339.Epub 2012 Jan 20, Baren JPet al.,Br J Cancer.2012 Jun 26;107(1):143-9.doi:10.1038 / bjc.2012.239.Epub 2012 Jun 7, Rao VHet al.,Oncogene.2012 Jun 7;31(23):2888-98.doi:10.1038 / onc.2011.460.Epub 2011 Oct 10, Kanakis D.et al.,Dis Markers.2013;34(2):81-91.doi:10.3233 / DMA-120953、Georges S.et al.,Eur J Cancer.2013 Jun;49(9):2253-63.doi:10.1016 / j.ejca.2013.02.020.Epub 2013 Mar 13、Cireap N.et al.,Pathol Oncol Res.2013 Oct;19(4):755-62.doi:10.1007 / s12253-013-9639-8.Epub 2013 May 6、Bilgin Dogru E.et al.,Tumour Biol.2014 Nov;35(11):11647-53.doi:10.1007 / s13277-014-2514-8.Epub 2014 Aug 20、Cheon D.J.Et al.,Carcinogenesis.2015 Jul;36(7):739-47.doi:10.1093 / carcin / bgv059.Epub 2015 Apr 29、Rao V.H.et al.,Mol Carcinog.2015 Oct;54(10):1026-36.doi:10.1002 / mc.22171.Epub 2014 May 5、Li Z.et al.,Oncol Rep.2015 Dec;34(6):3231-7、Liu G.et al.,Oncol Rep.2016 Nov;36(5):3005-3013.doi:10.3892 / or.2016.5064.Epub 2016 Sep 5、Frohlich C.et al.,Clin Cancer Res.2006 Dec 15;12(24):7359-68、Roy R.et al.,Mol Cancer Res.2017 Nov;15(11):1608-1622.doi:10.1158 / 1541-7786.MCR-17-0188.Epub 2017 Aug 1、Xiong L.et al.,J Proteomics.2018 Jun 30;182:34-44.doi:10.1016 / j.jprot.2018.04.033.Epub 2018 May 2、Veenstra V.L.et al.,Oncogenesis.2018 Nov 16;7(11):87.doi:10.1038 / s41389-018-0096-9). Thus, in some embodiments, the anti-ADAM12 agents of the present invention may bind to or target ADAM12 on cancer cells of the above-mentioned cancer types.

[0119] In some embodiments, the anti-ADAM12 agents of the present disclosure may bind to bladder cancer cells. In some embodiments, the anti-ADAM12 agents may bind to bone cancer cells. In some embodiments, the anti-ADAM12 agents may bind to brain cancer cells. In some embodiments, the anti-ADAM12 agents may bind to breast cancer cells. In some embodiments, the anti-ADAM12 agents may bind to colon cancer cells. In some embodiments, the anti-ADAM12 agents may bind to colorectal cancer cells. In some embodiments, the anti-ADAM12 agents may bind to desmoid tumor cells.

[0120] In some embodiments, the anti-ADAM12 agent may bind to esophageal cancer cells. In some embodiments, the anti-ADAM12 agent may bind to fibromatosis cells. In some embodiments, the anti-ADAM12 agent may bind to glioblastoma cells. In some embodiments, the anti-ADAM12 agent may bind to head and neck cancer cells. In some embodiments, the anti-ADAM12 agent may bind to liver cancer cells. In some embodiments, the anti-ADAM12 agent may bind to lung cancer cells. In some embodiments, the anti-ADAM12 agent may bind to melanoma cells. In some embodiments, the anti-ADAM12 agent may bind to esophagogastric adenocarcinoma cells.

[0121] In some embodiments, the anti-ADAM12 agent may bind to oligodendroglioma cells. In some embodiments, the anti-ADAM12 agent may bind to oral cancer cells. In some embodiments, the anti-ADAM12 agent may bind to oral squamous cell carcinoma cells. In some embodiments, the anti-ADAM12 agent may bind to osteosarcoma cells. In some embodiments, the anti-ADAM12 agent may bind to ovarian cancer cells. In some embodiments, the anti-ADAM12 agent may bind to pancreatic cancer cells. In some embodiments, the anti-ADAM12 agent may bind to prostate cancer cells. In some embodiments, the anti-ADAM12 agent may bind to skin cancer cells. In some embodiments, the anti-ADAM12 agent may bind to small cell lung cancer cells. In some embodiments, the anti-ADAM12 agent may bind to gastric cancer cells. In some embodiments, the anti-ADAM12 agent may bind to thyroid cancer cells.

[0122] Notably, ADAM12 overexpression in various diseases is in stark contrast to its expression on normal tissues, which is extremely low and limited to breast and ovarian tissues.In addition, ADAM12 expression is involved in accelerated tumor growth, promotion of tumor angiogenesis, and poor prognosis (Kveiborg M. et al.,Cancer Res.2005 Jun 1;65(11):4754-61;Roy R.et al.,Mol Cancer Res.2017 Nov;15(11):1608-1622.doi:10.1158 / 1541-7786.MCR-17-0188.Epub 2017 Aug 1).These observations particularly highlight the value of ADAM12 as a therapeutic target by this method.

[0123] ADAM12 is also upregulated and / or plays a pathological role in many other diseases and conditions, including, but not limited to, Alzheimer's disease, osteoarthritis, muscular dystrophy, multiple sclerosis, fibrosis, cardiac hypertrophy, dermal fibrosis and interstitial lung disease in systemic sclerosis, renal fibrosis, peripheral arterial disease, endometriosis, and Dupuytren's disease (Kveiborg M. et al., Int J Biochem Cell Biol. 2008;40(9):1685-702. doi:10.1016 / j.biocel.2008.01.025. Epub 2008 Feb 1; Harold D. et al., Am J Med Genet B Neuropsychiatr Genet. 2007 Jun 5;144B(4):448-52; Kerna I. et al., Rheumatol Int. 2012 Feb;32(2):519-23.doi:10.1007 / s00296-010-1717-6.Epub 2011 Jan 22, Dulauroy S.et al.,Nat Med.2012 Aug;18(8):1262-70.doi:10.1038 / nm.2848.Epub 2012 Jul 29, Berry E.et al.,J Vasc Res.2013;50(1):52-68.doi:10.1159 / 000345240.Epub 2012 Nov 17, Taniguchi T.et al.,J Eur Acad Dermatol Venereol.2013 Jun;27(6):747-53.doi:10.1111 / j.1468-3083.2012.04558.x.Epub 2012 Apr 28, Ramdas V.et al.,Am J Pathol.2013 Dec;183(6):1885-1896.doi:10.1016 / j.ajpath.2013.08.027.Epub 2013 Oct 6, Dokun AOet al.,Am J Physiol Heart Circ Physiol.2015 Sep;309(5):H790-803.doi:10.1152 / ajpheart.00803.2014.Epub 2015 Jul 10, Miller MAet al.,Sci Rep.2015 Oct 19;5:15150.doi:10.1038 / srep15150; Sedic M. et al.,(2012) Using Functional Genomics to Identify Drug Targets:A Dupuytren's Disease Example.In:Larson R.(ed.) Bioinformatics and Drug Discovery.Methods in Molecular Biology(Methods and Protocols),vol 910.Humana Press,Totowa,NJ).Thus, in some embodiments, the anti-ADAM12 agents of the present invention may bind to or target ADAM12 on cells of the above-mentioned diseases or conditions.

[0124] In some embodiments, the anti-ADAM12 agent or composition of the present disclosure may be used to treat Alzheimer's disease. In some embodiments, the anti-ADAM12 agent or composition may be used to treat osteoarthritis. In some embodiments, the anti-ADAM12 agent or composition may be used to treat muscular dystrophy. In some embodiments, the anti-ADAM12 agent or composition may be used to treat multiple sclerosis. In some embodiments, the anti-ADAM12 agent or composition may be used to treat fibrosis. In some embodiments, the anti-ADAM12 agent or composition may be used to treat cardiac hypertrophy. In some embodiments, the anti-ADAM12 agent or composition may be used to treat dermal fibrosis and interstitial lung disease in systemic sclerosis. In some embodiments, the anti-ADAM12 agent or composition may be used to treat renal fibrosis. In some embodiments, the anti-ADAM12 agent or composition may be used to treat peripheral arterial disease. In some embodiments, the anti-ADAM12 agent or composition may be used to treat endometriosis. In some embodiments, an anti-ADAM12 agent or composition may be used to treat Dupuytren's disease.

[0125] Anti-ADAM12 antibodies, antigen-binding fragments, multispecific antibodies, multispecific antigen-binding fragments, and antibody-drug conjugates In some embodiments, the anti-ADAM12 antibodies (Abs), anti-ADAM12 antigen-binding (AB) fragments, anti-ADAM12 multispecific Abs, anti-ADAM12 multispecific antigen-binding Ab fragments, and anti-ADAM12 antibody-drug conjugates (ADCs) of the present invention each comprise at least one AB domain that binds to ADAM12.

[0126] The ADAM12 binding domain (i.e., the AB domain) may comprise the AB domain of a humanized version of a mouse anti-ADAM12 monoclonal antibody. In some embodiments, the ADAM12 binding domain (i.e., the AB domain) may comprise the AB domain of a humanized version of the mouse anti-ADAM12 monoclonal antibody clones 6E6 and 6C10 or variants thereof.

[0127] The murine anti-ADAM12 monoclonal antibody 6E6 comprises (a) a heavy chain (HC) variable domain (VH) sequence set forth in SEQ ID NO: 111, which may be encoded by SEQ ID NO: 211, and (b) a light chain (LC) variable domain (VL) sequence set forth in SEQ ID NO: 115, which may be encoded by SEQ ID NO: 215. Complementarity determining regions 1, 2, and 3 (CDR1, CDR 2, and CDR 3) of VH (i.e., CDR-H1, CDR-H2, and CDR-H3) comprise the amino acid sequences of SEQ ID NOs: 112, 113, and 114, respectively, which may be encoded by SEQ ID NOs: 212, 213, and 214, respectively. CDR1, CDR2, and CDR3 of VL (i.e., CDR-L1, CDR-L2, and CDR-L3) comprise the amino acid sequences of SEQ ID NOs: 116, 117, and 118, respectively, which can be encoded by SEQ ID NOs: 216, 217, and 218, respectively.

[0128] The mouse anti-ADAM12 monoclonal antibody clone 6C10 comprises (a) a VH sequence as set forth in SEQ ID NO: 121, which can be encoded by SEQ ID NO: 221, and (b) a VL sequence as set forth in SEQ ID NO: 125, which can be encoded by SEQ ID NO: 225. The CDR1, CDR2, and CDR3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) comprise the amino acid sequences of SEQ ID NOs: 122, 123, and 124, respectively, which can be encoded by SEQ ID NOs: 222, 223, and 224, respectively. The CDR1, CDR2, and CDR3 of the VL (i.e., CDR-L1, CDR-L2, and CDR-L3) comprise the amino acid sequences of SEQ ID NOs: 126, 127, and 128, respectively, which can be encoded by SEQ ID NOs: 226, 227, and 228, respectively.

[0129] The inventors performed humanization of the 6E6 antibody as described in Example 1 herein. The humanized embodiment of 6E6 (which may be referred to as h6E6) comprises (a) the VH sequence set forth in SEQ ID NO: 131, which may be encoded by SEQ ID NO: 231, and (b) the VL sequence set forth in SEQ ID NO: 135, which may be encoded by SEQ ID NO: 235. The CDR 1, CDR 2, and CDR 3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino acid sequences of SEQ ID NOs: 132, 133, and 134, respectively, which may be encoded by SEQ ID NOs: 232, 233, and 234, respectively. The CDR 1, CDR 2, and CDR 3 of the VL (i.e., CDR-L1, CDR-L2, and CDR-L3) may comprise the amino acid sequences of SEQ ID NOs: 136, 137, and 138, respectively, which may be encoded by SEQ ID NOs: 236, 237, and 238, respectively.

[0130] The inventors have performed humanization of the 6C10 antibody as described in Example 1 herein. The humanized embodiment of 6C10 (which may be referred to as h6C10) comprises (a) a VH sequence set forth in SEQ ID NO: 141, which may be encoded by SEQ ID NO: 241, and (b) a VL sequence set forth in SEQ ID NO: 145, which may be encoded by SEQ ID NO: 245. CDR 1, CDR 2, and CDR 3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino acid sequences of SEQ ID NOs: 142, 143, and 144, respectively, which may be encoded by SEQ ID NOs: 242, 243, and 244, respectively. CDR1, CDR2, and CDR3 of the VL (i.e., CDR-L1, CDR-L2, and CDR-L3) can comprise the amino acid sequences of SEQ ID NOs: 146, 147, and 148, respectively, which can be encoded by SEQ ID NOs: 246, 247, and 248, respectively.

[0131] Thus, in some embodiments, the ADAM12 binding domain (i.e., the AB domain) of an anti-ADAM12 agent of the present invention may comprise (a) CDR-H1, CDR-H2, and CDR-H3, and (b) CDR-L1, CDR-L2, and CDR-L3.

[0132] In some embodiments, the ADAM12 binding domain (i.e., the AB domain) may comprise (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3, and a human-like HC framework, and (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3, and a human-like LC framework.

[0133] In some embodiments, in the AB binding domain of the anti-ADAM12 agent of the invention, CDR-H1, CDR-H2, and CDR-H3 may comprise the amino acid sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively, and CDR-L1, CDR-L2, and CDR-L3 may comprise the amino acid sequences set forth in SEQ ID NOs: 136, 137, and 138, respectively. CDR-H1, CDR-H2, and CDR-H3 may comprise the amino acid sequences encoded by SEQ ID NOs: 232, 233, and 234, respectively, and CDR-L1, CDR-L2, and CDR-L3 may comprise the amino acid sequences encoded by SEQ ID NOs: 236, 237, and 238, respectively.

[0134] In some embodiments, CDR-H1, CDR-H2, and CDR-H3 may comprise the amino acid sequences set forth in SEQ ID NOs: 142, 143, and 144, respectively, and CDR-L1, CDR-L2, and CDR-L3 may comprise the amino acid sequences set forth in SEQ ID NOs: 146, 147, and 148, respectively. CDR-H1, CDR-H2, and CDR-H3 may comprise the amino acid sequences encoded by SEQ ID NOs: 242, 243, and 244, respectively, and CDR-L1, CDR-L2, and CDR-L3 may comprise the amino acid sequences encoded by SEQ ID NOs: 246, 247, and 248, respectively.

[0135] Generally, to humanize a mouse antibody, CDRs from the mouse antibody may be grafted onto a human antibody framework. Thus, the human-like framework may be 100% identical to the human framework. The inventors used the Tabhu program (http: / / circe.med.uniroma1.it / tabhu / ) to perform the humanization, which includes four steps: (i) loop grafting, (ii) estimation of binding mode similarity between natural and human antibodies, (iii) back mutation, and (iv) re-evaluation of binding mode similarity between the input antibody and the humanized antibody (Olimpieri PP et al., Bioinformatics. 2015 Feb 1;31(3):434-5. doi:10.1093 / bioinformatics / btu667. Epub 2014 Oct 9). Thus, in some embodiments, the framework may not be 100% identical to the human framework, but may still contain significant sequence identity to the human framework.

[0136] In some embodiments, (a) the human-like HC framework may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human HC framework; and (b) the human-like LC framework may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human LC framework.

[0137] In some embodiments, the variable domain of the anti-ADAM12 agent disclosed herein may be altered without inhibiting ADAM12 binding. The sequence of the variable domain may be altered as long as the AB domain binds sufficiently to ADAM12. The antigen-Ab interaction is determined primarily by the six CDRs, but those skilled in the art will understand that some deviation from the exact CDR sequence may be possible. Any suitable technique, such as affinity maturation, may be used to alter the CDR sequence. Of the six CDRs, VH CDR 3 and VL CDR 3 are generally considered to be the major determinants of specificity in antigen recognition. In particular, diversity in CDR 3 of VH (i.e., CDR-H3) may be particularly important to provide the most antibody specificity (Xu JL, Immunity. 2000 Jul; 13(1): 37-45). Thus, one or more mutations may be incorporated into CDR 1 and / or CDR 2 without significantly reducing binding affinity while achieving more desired properties of the Ab. Also within the scope of the present invention are Abs or antigen-binding Ab fragments comprising CDR-H1, CDR-H2, CDR-L1, CDR-L2, and / or CDR-L3 that are at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CDR-H1, CDR-H2, CDR-L1, CDR-L2, and / or CDR-L3 disclosed herein, respectively. Additionally, one or more mutations in CDR-H3 may be incorporated to alter, increase, or fine-tune the binding or any other property of the AB domain.Alternatively, since any one mutation may alter biochemical properties such as thermodynamic stability or immunogenicity in addition to affinity, one may test all possible mutations in any of the six CDRs, and / or any combination thereof, and / or even in the framework sequences to see if the sequence modification provides improved or more desirable overall properties (Rajpal A. et al., Proc Natl Acad Sci US A. 2005 Jun 14; 102(24):8466-8471.doi:10.1073 / pnas.0503543102; Julian MCet al.,Sci Rep.2017;7:45259.Prepublished online March 28, 2017,doi:10.1038 / srep45259). Any suitable technique may be used to test binding of the modified Ab to the target, including but not limited to, ELISA, RIA, FACS, bioassay, or Western blot assay.

[0138] In some embodiments, the HC variable domain (i.e., VH) may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:131, and the LC variable domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:135. The HC variable domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:231, and the LC variable domain (i.e., VL) comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:235.

[0139] In some embodiments, the VH may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:141, and the VL may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:145. The VH may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:241, and the VL may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:245.

[0140] In some embodiments, the anti-ADAM12 agents of the present invention can be, for example, but not limited to, monoclonal Abs, monospecific Abs, bispecific Abs, multispecific Abs, humanized Abs, tetrameric Abs, tetravalent Abs, single chain Abs, domain specific Abs, domain deleted Abs, scFc fusion proteins, chimeric Abs, synthetic Abs, recombinant Abs, hybrid Abs, mutated Abs, CDR grafted Abs, fragment antigen binding (Fab), F(ab')2, Fab' fragment, variable fragment (Fv), single chain Fv (scFv) fragment, Fd fragment, diabody, and minibody.

[0141] In certain embodiments, the AB domain of an anti-ADAM12 agent of the present invention may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by (i) SEQ ID NO: 139, 140, 149, or 150, or (ii) SEQ ID NO: 239, 240, 249, or 250.

[0142] In some embodiments, an anti-ADAM12 agent of the invention may comprise two or more binding specificities (i.e., bispecific, trispecific, or generally multispecific). The first specificity is for an epitope of ADAM12 (the first ADAM12 epitope).

[0143] In one embodiment, an anti-ADAM12 agent of the present disclosure may have a second binding specificity for another epitope of ADAM12 (i.e., a second ADAM12 epitope). The second ADAM12 epitope may or may not overlap with the first ADAM12 epitope.

[0144] In another aspect, the second specificity can be specificity for an epitope of a second antigen other than ADAM12. The multispecific ADAM12 binding agent of the present disclosure can bind to ADAM12 and one or more other targets. In some embodiments, the multispecific anti-ADAM12 agent binds to ADAM12 and proteins on effector cells. In some embodiments, the multispecific anti-ADAM12 agent binds to ADAM12 and proteins on target (e.g., cancer) cells. In some embodiments, binding to the second antigen can improve the functional characteristics of the anti-ADAM12 agent, such as recruitment, effector function, lysis of target cells.

[0145] In certain embodiments, the second antigen can be, for example, but not limited to, CD3, NKG2D, 4-1BB, or an Fc receptor (FcR), such as Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIB1, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha / mu receptor (Fca / mR), or neonatal Fc receptor (FcRn). For anti-ADAM12 Abs and antigen-binding Ab fragments, the specificity for FcR allows for FcR-mediated effects, such as antibody-dependent cellular phagocytosis (ADCP) or antibody-dependent cellular cytotoxicity (ADCC) of ADAM12-expressing cells, or release of cytotoxic mediators by Fc-expressing cells.

[0146] If the second epitope is within an FcR, the FcR can be, but is not limited to, an Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIB1, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha / mu receptor (Fca / mR), or neonatal Fc receptor (FcRn).

[0147] When an anti-ADAM12 agent has two specificities, the agent can be called bispecific. Bispecific anti-ADAM12 agents include bispecific anti-ADAM12 Ab or antigen-binding Ab fragments. When an anti-ADAM agent has two or more specificities, the agent can be called multispecific. Multispecific anti-ADAM12 agents include multispecific anti-ADAM12 Ab or antigen-binding Ab fragments.

[0148] The invention encompasses any type of bispecific Ab-like molecule (Ab or antigen-binding Ab fragment) as outlined in Brinkmann U. et al., MAbs. 2017 Feb-Mar; 9(2): 182-212. Prepublished online Jan. 10, 2017, doi: 10.1080 / 19420862.2016.1268307, Klein C. et al., MAbs. 2016 Aug-Sep; 8(6): 1010-20. doi: 10.1080 / 19420862.2016.1197457. In bispecific embodiments of the disclosure, one of the AB domains is an anti-ADAM12 binding domain. General methods for designing and constructing bispecific or multispecific Ab or antigen-binding Ab fragments are known in the art (Brinkmann U. et al., MAbs. 2017 Feb-Mar; 9(2): 182-212. Prepublished online January 10, 2017, doi: 10.1080 / 19420862.2016.1268307; Dimasi N. et al. Methods. 2018 Aug 11. pii: S1046-2023(18)30149-X. doi: 10.1016 / j.ymeth.2018.08.004; Sedykh SE et al., Drug Des Devel Ther. 2018; 12: 195-208). Such methods include chemical conjugation, covalent linkage of fragments, and genetic engineering. For example, full-length bispecific Ab or antigen-binding Ab fragments can be produced by co-expressing two pairs of heavy and light chains, each with a different specificity. The two pairs can be encoded in one vector or in separate vectors, but expressed in the same host cell (hsot cell). Alternatively, antigen-binding Ab fragments or AB domains with different specificities can be produced separately and then conjugated to each other, for example, by sulfhydryl binding (e.g., binding of the C-terminal hinge region of HC) and / or using a suitable coupling or cross-linking agent.Bispecific antigen-binding Ab fragments may also be generated, for example, by using leucine zippers or by using scFv dimers (see, for example, Kosteln et al., J Immunol. 1992 Mar 1;148(5):1547-53). Binding of the bispecific agents of the invention may be confirmed using any suitable method, such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, bioassay, or Western blot.

[0149] In some embodiments, an anti-ADAM12 agent of the invention may comprise a human-like fragment crystallizable (Fc) region.

[0150] In some embodiments, the human-like Fc region can be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region.

[0151] In certain embodiments, the human-like Fc region can bind to an Fc receptor (FcR), which can be, for example, but not limited to, an Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIB1, FcgRIIB2, FcgRIIIA, FcgRIIIB, an Fc epsilon receptor (FceR), FceRI, FceRII, an Fc alpha receptor (FcaR), FcaRI, an Fc alpha / mu receptor (Fca / mR), or a neonatal Fc receptor (FcRn).

[0152] In some embodiments, when the anti-ADAM12 agent is an Ab, the Ab can be of the IgM, IgD, IgG, IgE, or IgA isotype.

[0153] In some embodiments, when the Ab is an IgG, the IgG can be an IgG1, IgG2, IgG3, or IgG4.

[0154] Certain amino acid modifications in the Fc region are known to modulate Ab effector functions and properties, including but not limited to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and half-life (Wang X. et al., Protein Cell. 2018 Jan;9(1):63-73; Dall'Acqua WF et al., J Biol Chem. 2006 Aug 18;281(33):23514-24. Epub 2006 Jun 21; Monnet C. et al, Front Immunol. 2015 Feb 4;6:39. doi:10.3389 / fimmu.2015.00039. eCollection 2015). Mutations can be symmetric or asymmetric. In certain cases, antibodies with Fc regions with asymmetric mutations (i.e., the two Fc regions are not identical) may confer better functionality, such as ADCC (Liu Z. et al. J Biol Chem. 2014 Feb 7;289(6):3571-3590).

[0155] If the Ab is an IgG1, the Fc region may contain one or more amino acid substitutions, such as, for example, N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236 deletion, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300 L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270 E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y The Fc region may further comprise one or more additional amino acid substitutions. The substitutions may be, for example, but are not limited to, A330L, L234F, L235E, P3318, and / or any combination thereof (residue numbering according to EU or Kabat numbering).

[0156] When the Ab is an IgG2, the Fc region may include one or more amino acid substitutions. The substitutions may be, for example, but are not limited to, P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and / or any combination thereof (residue numbering according to EU or Kabat numbering). The Fc region may further include one or more additional amino acid substitutions. The substitutions may be, for example, but are not limited to, M252Y, S254T, T256E, and / or any combination thereof (residue numbering according to EU or Kabat numbering).

[0157] If the Ab is an IgG3, the Fc region may contain one or more amino acid substitutions. The substitution may be, for example, but is not limited to, E235Y (residue numbering according to EU or Kabat numbering).

[0158] When the Ab is IgG4, the Fc region may contain one or more amino acid substitutions. Substitutions may be, for example, but not limited to, E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and / or any combination thereof (residue numbering according to EU or Kabat numbering). Substitutions may be, for example, S228P (residue numbering according to EU or Kabat numbering).

[0159] In some embodiments, the glycans of the human-like Fc region can be engineered to alter effector function (see, e.g., Li T. et al., Proc Natl Acad Sci US A. 2017 Mar 28; 114(13): 3485-3490. doi: 10.1073 / pnas.1702173114. Epub 2017 Mar 13).

[0160] In some embodiments, an anti-ADAM12 agent of the invention may be an antibody-drug conjugate (ADC). The ADC may comprise (a) any Ab or antigen-binding Ab fragment described herein, and (b) a drug conjugated to the Ab or antigen-binding Ab fragment.

[0161] In some embodiments, the drug can be, but is not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an antibacterial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an antifibrotic drug, an immunosuppressant drug, a steroid, a bronchodilator, a beta-blocker, a matrix metalloproteinase inhibitor, an ADAM12 inhibitor, an ADAM12 signaling inhibitor, an enzyme, a hormone, a neurotransmitter, a toxin, a radioisotope, a chemical compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, a shRNA, a microRNA, an oligonucleotide, and an imaging drug.

[0162] The toxin may be a bacterial, fungal, plant or animal toxin, or a fragment thereof. Examples include, but are not limited to, diphtheria A chain, diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alphasarcin, Aleurites fordii protein, dianthin protein, or Phytolacca Americana protein.

[0163] Anticancer or antiproliferative drugs include, but are not limited to, doxorubicin, daunorubicin, cucurbitacin, chaetocin, chaetoglobocin, chlamydocin, calicheamicin, nemorubicin, cryptophycin, menthacarcin, ansamitocin, mitomycin C, geldanamycin, mecalcarmycin, rebeccamycin, safracin, oxilactomycin, oligomycin, actinomycin, sandramycin, hypothemicin, , polyketomycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, taltoburine, lactacystin, dolastatin, auristatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), telomestatin, tubastatin A, combretastatin, maytansinoids, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodiazepines, SN-38, Ro 5-3335, pwainaphycin, duocarmycin, bafilomycin, taxoid, tubulysin, ferulenol, luciol A, fumagillin, hygrolysin, glucopyericidin, amanitin, ansatrienin, sinerubin, phallacidin, phalloidin, phytosphongosine, piericidin, poronetin, phodophyllotoxin, gramicidin A, sanguinarine, sinefungin, herboxidiene, microcorrin B, microcystin, muscotoxin A , trytoxin, trypolin A, myoseverin, mitoxin B, nocuolin A, pseudodoralic acid B, pseudolotin A, cyclopamine, curbulin, colchicine, aphidicolin, englerin, cordycepin, apoptolidin, epothilone A, limaquinone, isatropolone, isofistularin, quinaldopeptin, ixabepilone, aeroprisinin, arruginosin, agrochelin, epothilone, and derivatives thereof (see, for example, Polakis P. et al., Pharmacol Rev.2016 Jan;68(1):3-19. doi:10.1124 / pr.114.009373) (drugs may be obtained from a number of vendors, including Creative Biolabs®).

[0164] The radioisotope can be, for example, but not limited to, At211, I131, In131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and a radioisotope of Lu.

[0165] In certain embodiments, the drug can be, but is not limited to, MMAE or MMAF.

[0166] In some embodiments, the Ab or antigen-binding Ab fragment is directly conjugated to a drug to form an ADC.

[0167] In some embodiments, the Ab or antigen-binding Ab fragment is indirectly conjugated to a drug to form an ADC.

[0168] Any suitable conjugation method may be used to generate the ADC (e.g., Nolting B. Methods Mol Biol. 2013;1045:71-100. doi:10.1007 / 978-1-62703-541-5_5; Jain N. et al., Pharm Res. 2015 Nov;32(11):3526-40. doi:10.1007 / s11095-015-1657-7. Epub 2015 Mar 11; Tsusuchikama K. et al., Protein Cell. 2018 Jan;9(1):33-46. doi:10.1007 / s13238-016-0323-0. Epub 2016 Oct 14; Polakis P. et al., Pharmacol Rev. 2016 Jan;68(1):3-19.doi:10.1124 / pr.114.009373). Examples of methods that can be used to carry out conjugation include, but are not limited to, chemical conjugation and enzymatic conjugation.

[0169] Chemical conjugation may utilize, for example, but is not limited to, lysine amide coupling, cysteine ​​coupling, and / or incorporation of unnatural amino acids by genetic engineering. Enzymatic conjugation may utilize, for example, but is not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and / or N-glycan engineering.

[0170] In certain embodiments, one or more of the cleavable linkers can be used for conjugation. The cleavable linker can be, for example, but not limited to, responsive to the environmental difference between the extracellular and intracellular environment (pH, redox potential, etc.) or allow the cleavage of the drug by a specific lysosomal enzyme.

[0171] Examples of cleavable linkers include, but are not limited to, hydrazone linkers, peptide linkers including cathepsin B-responsive linkers, e.g., valine-citrulline (vc) linkers, disulfide linkers, e.g., N-succinimidyl-4-(2-pyridyldithio) (SPP) linker, or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) linker, and pyrophosphate diester linkers are included.

[0172] Alternatively, or simultaneously, one or more of the non-cleavable linkers can be used.Examples of non-cleavable linkers include thioether linkers such as N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and maleimidocaproyl (mc) linkers.In general, non-cleavable linkers are more resistant to proteolysis and more stable than cleavable linkers.

[0173] Anti-ADAM12 chimeric antigen receptor (CAR) In some embodiments, the anti-ADAM12 agent of the present disclosure can be a chimeric antigen receptor (CAR). In particular, the CAR of the present invention comprises an antigen binding (AB) domain that binds to ADAM12, a transmembrane (TM) domain, and an intracellular signaling (ICS) domain.

[0174] A schematic diagram showing a general CAR construct of the invention is shown in FIG. 1A.

[0175] The CAR may optionally comprise a hinge connecting the AB domain and the above-mentioned TM domain.

[0176] The CAR may optionally contain one or more costimulatory (CS) domains.

[0177] Schematic diagrams showing two or more typical CAR constructs of the invention are shown in Figures 1B and 1C.

[0178] AB domain The CAR of the present invention comprises an antigen-binding (AB) domain that binds to ADAM12.

[0179] In some embodiments, the AB domain of the CAR may comprise any of the anti-ADAM12 agents disclosed herein.

[0180] In some embodiments, the AB domain of the CAR may comprise any of the AB domains of any of the anti-ADAM12 agents disclosed herein.

[0181] In some embodiments, the AB domain of the CAR may comprise any of the anti-ADAM12 Abs, anti-ADAM12 antigen-binding Ab fragments, anti-ADAM12 multispecific Abs, anti-ADAM12 multispecific antigen-binding Ab fragments, and anti-ADAM12 ADCs, or AB domains thereof, disclosed herein.

[0182] In some embodiments, the AB domain of the CAR may comprise an anti-ADAM12 scFv.

[0183] In some embodiments, the AB domain may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by (i) SEQ ID NO: 139, 140, 149, or 150, or (ii) SEQ ID NO: 239, 240, 249, or 250.

[0184] In some embodiments, the AB domain can compete for binding to ADAM12 with an scFv comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by (i) SEQ ID NO: 139, 140, 149, or 150, or (ii) SEQ ID NO: 239, 240, 249, or 250.

[0185] Alternatively, or concurrently, the AB domain may comprise an ADAM12-binding portion of a molecule that binds to ADAM12.

[0186] Examples of physiological ADAM12 substrates include, but are not limited to, alpha actinin 2 (ACTN2), insulin-like growth factor binding protein 3 (IGFBP3), IGFBP-5, phosphatidylinositol 3 kinase regulatory subunit alpha (PIK3R1), heparin-binding epidermal growth factor (HB-EGF), epidermal growth factor (EGF), betacellulin, delta-like 1, and placental leucine aminopeptidase (P-LAP), as well as matrix metalloproteases (MMP-14) (Galliano MF et al., J Biol Chem. 2000 May 5; 275(18): 13933-9; Kveiborg M. et al., Int J Biochem Cell Biol. 2008; 40(9): 1685-702. doi: 10.1016 / j.biocel.2008.01.025. Epub 2008 Feb 1; Kang Q.et al., J Biol Chem.2001 Jul 6;276(27):24466-72.Epub 2001 Apr 19; Albrechtsen R.et al., J Cell Sci.2013 Oct 15;126(Pt 20):4707-20.doi:10.1242 / jcs.129510.Epub 2013 Sep 4). The amino acid sequences of the above-mentioned ADAM12 substrates are set forth in SEQ ID NOs: 151, 152, 153, 154, 155, 156, 157, 158, and 159, respectively.

[0187] An example of the AB domain of the CAR of the present invention, or any other anti-ADAM12 agent, is shown in FIG.

[0188] Hinge In some embodiments, CAR may comprise a hinge sequence between the AB domain and the TM domain. Those skilled in the art will understand that a hinge sequence is a short sequence of amino acids that promotes flexibility (see, for example, Woof JMet al., Nat. Rev. Immunol., 4(2):89-99 (2004)). The hinge sequence may be any suitable sequence derived from or obtained from any suitable molecule.

[0189] In some embodiments, the length of the hinge sequence may be optimized based on the desired length of the extracellular portion of the CAR, which may be based on the location of the epitope within the target molecule. For example, if the epitope is in the membrane proximal region within the target molecule, a longer hinge may be optimal.

[0190] In some embodiments, the hinge may be derived from or include at least a portion of an immunoglobulin Fc region, such as an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the hinge includes at least a portion of an IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains. In some embodiments, the hinge may also include at least a portion of a corresponding immunoglobulin hinge region. In some embodiments, the hinge is derived from or includes at least a portion of a modified immunoglobulin Fc region, such as an modified IgG1 Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region. The modified immunoglobulin Fc region may have one or more mutations (e.g., point mutations, insertions, deletions, duplications) that result in one or more amino acid substitutions, modifications, or deletions that result in impaired binding of the hinge to an Fc receptor (FcR). In some embodiments, the modified immunoglobulin Fc region may be designed with one or more mutations that result in one or more amino acid substitutions, modifications, or deletions that result in impaired binding of the hinge to one or more FcRs, including, but not limited to, FcγRI, FcγR2A, FcγR2B1, Fcγ2B2, Fcγ3A, Fcγ3B, FcεRI, FcεR2, FcαRI, Fcα / μR, or FcRn.

[0191] In some embodiments, a portion of the immunoglobulin constant region functions as a hinge between the AB domain (e.g., scFv or nanobody) and the TM domain. The hinge can be of a length that results in increased responsiveness of the CAR-expressing cell after antigen binding compared to the absence of the hinge. In some examples, the hinge is 12 amino acids long or about 12 amino acids long, or is 12 amino acids long or less. Exemplary hinges include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, and including any integer between any endpoints of the recited ranges. In some embodiments, the hinge has about 12 or less amino acids, about 119 or less amino acids, or about 229 or less amino acids.Exemplary hinges include CD28 hinges, IgG4 hinges alone, IgG4 hinges attached to CH2 and CH3 domains, or IgG4 hinges attached to CH3 domains.Exemplary hinges include, but are not limited to, those described in Hudecek M.et al.(2013)Clin.Cancer Res.,19:3153, International Patent Application Publication No. WO2014 / 031687, U.S. Patent No. 8,822,647, or published application No. US2014 / 0271635.

[0192] In some embodiments, the hinge sequence is derived from a CD8α molecule or a CD28 molecule. In preferred embodiments, the hinge sequence is derived from CD28. In one embodiment, the hinge comprises the amino acid sequence of a human CD28 hinge (SEQ ID NO: 163) or a sequence encoded by SEQ ID NO: 263. In some embodiments, the hinge has an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 163.

[0193] Transmembrane (TM) domain Regarding the TM domain, the CAR can be designed to include a TM domain that is fused with the AB domain of the CAR. A hinge sequence can be inserted between the AB domain and the TM domain. In one embodiment, the TM domain that naturally associates with one of the domains in the CAR is used. In some cases, the TM domain can be selected or modified by amino acid substitution to avoid the binding of such domains with the transmembrane domain of the same or different surface membrane protein, thereby minimizing the interaction with other members of the receptor complex.

[0194] TM domains can be derived from either natural or synthetic sources. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Typically, a TM domain represents a single transmembrane alpha-helix of a transmembrane protein, also known as an integral protein. TM domains of particular use in the present invention can be derived from TM domains including CD28, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCR alpha, TCR beta, or CD3 zeta and / or functional variants thereof, e.g., those that retain a significant portion of their structural (e.g., transmembrane) properties (i.e., may include their transmembrane regions).

[0195] Alternatively, the TM domain may be synthetic, in which case it will mainly comprise hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic TM domain. The TM domain of the present invention is thermodynamically stable in membranes. It may be a single α-helix, a transmembrane β-barrel, a gramicidin A β-helix, or any other structure. The transmembrane helix is ​​usually about 20 amino acids long.

[0196] Preferably, the TM domain in the CAR of the invention is derived from the TM region of CD28. In one embodiment, the TM domain comprises the amino acid sequence of human CD28 TM (SEQ ID NO: 161), or the sequence encoded by SEQ ID NO: 261. In some embodiments, the TM domain comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 161.

[0197] Optionally, a short oligo- or polypeptide spacer, preferably 2-10 amino acids in length, may form the link between the TM domain and the ICS domain of the CAR. A glycine-serine doublet may provide a suitable spacer.

[0198] Intracellular signaling (ICS) and costimulatory (CS) domains The ICS domain or other cytoplasmic domain of the CAR of the present invention triggers or induces the activation of at least one of the normal effector functions of the cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. 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" or "ICS 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 ICS 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, it can be used in place of the intact chain, so long as such a truncated portion transmits the effector function signal. Thus, the term "intracellular signaling domain" or "ICS domain" is meant to include any truncated portion of the ICS domain that is sufficient to transmit the effector function signal.

[0199] Preferred examples of ICS domains for use in the CARs of the present invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capability.

[0200] The signal generated through only one ICS domain may be insufficient for the complete activation of the cell, and a secondary or costimulatory signal may also be required. In such cases, a costimulatory domain (CS domain) may be included in the cytoplasmic part of the CAR. The CS domain is a domain that transduces such a secondary or costimulatory signal. Optionally, the CAR of the present invention may contain two or more CS domains. The CS domain may be located upstream of the ICS domain or downstream of the ICS domain. Two exemplary schematic diagrams of the CAR construct of the present invention containing at least one CS domain are shown in Figure 1B and Figure 1C.

[0201] In some embodiments, T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner can include signaling motifs known as immunoreceptor tyrosine-dependent activation motifs or ITAMs. Such cytoplasmic signaling sequences can be included in the ICS or CS domains of the CAR of the present invention.

[0202] Examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly useful in the present invention include those derived from the ICS domain of the lymphocyte receptor chain, the TCR / CD3 complex protein, the Fc receptor subunit, the IL-2 receptor subunit, CD3zeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, and DAP12.

[0203] It is particularly preferred that the ICS domain in the CAR of the present invention comprises a cytoplasmic signaling sequence derived from CD3 zeta. In one embodiment, the ICS domain comprises the amino acid sequence of human CD3ζICS (SEQ ID NO: 162), or the sequence encoded by SEQ ID NO: 262. In some embodiments, the ICS domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 162.

[0204] In a preferred embodiment, the cytoplasmic domain of the CAR can be designed to contain the CD3ζ ICS domain by itself. In another preferred embodiment, the CD3ζ ICS domain can be combined with one or more of any other desired cytoplasmic domains useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of the CAR can contain the CD3ζ ICS domain and the CS domain. The CS region refers to the part of the CAR that contains the intracellular domain of the costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for the efficient response of lymphocytes to antigens.

[0205] Various CS domains have been reported to confer different properties. For example, the 4-1BB CS domain showed enhanced persistence in in vivo xenograft models (Milone MC et al. Mol Ther 2009;17:1453-1464, Song DGet al. Cancer Res 2011;71:4617-4627). In addition, these different CS domains may generate different cytokine profiles, which in turn generate target cell-mediated cytotoxicity and effects on the disease microenvironment. Indeed, DAP10 signaling in NK cells has been associated with an increase in Th1 and inhibition of Th2-type cytokine production in CD8+ T cells (Barber A. et al. Blood 2011;117:6571-6581).

[0206] Examples of costimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, Toll ligand receptors, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5, CD7, CD8 α, CD8 β, CD11a, LFA-1 (CD11a / CD18), CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, CRTAM, OX40 (CD134), 4-1BB (C D137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF 4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, IL2R β, IL2R γ, IL7R α, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, ​​NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, ligands that specifically bind to CD83, etc. Thus, although the present invention is primarily exemplified by the regions of CD28, DAP10, and / or 4-1BB as the CS domain, other costimulatory elements are within the scope of the present invention.

[0207] The ICS and CS domains of the CAR of the invention may be linked together in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the linker. A glycine-serine doublet provides a particularly suitable linker.

[0208] In one embodiment, the CAR is designed to include a cytoplasmic signaling sequence of CD3ζ as the ICS domain and a cytoplasmic signaling sequence of CD28 as the CS domain. In another embodiment, the CAR is designed to include a cytoplasmic signaling sequence of CD3ζ as the ICS domain and a cytoplasmic signaling sequence of DAP10 as the CS domain. In yet another embodiment, the CAR is designed to include a cytoplasmic signaling sequence of CD3ζ as the ICS domain and a cytoplasmic signaling sequence of 4-1BB as the CS domain. Such a cytoplasmic signaling sequence of CD3ζ can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CD3ζ ICS domain comprising the amino acid sequence of human CD3zICS (SEQ ID NO: 162). Such cytoplasmic signaling sequences of CD3 zeta can be encoded by a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:262.

[0209] Such cytoplasmic signaling sequences of CD28 can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the human CD28 CS domain (SEQ ID NO: 164). Such cytoplasmic signaling sequences of CD28 can be encoded by a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:264. Such cytoplasmic signaling sequences of DAP10 can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the human 4-1BB CS domain (SEQ ID NO: 165). Such cytoplasmic signaling sequences of 4-1BB can be encoded by a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:265. Such cytoplasmic signaling sequences of DAP10 can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the human DAP10 CS domain (SEQ ID NO: 166). Such cytoplasmic signaling sequences of DAP10 can be encoded by a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:266.

[0210] Alternatively, if the AB domain comprises an ADAM12-binding portion of a molecule that binds to ADAM12 as described above, the TM domain of the CAR can be derived from the transmembrane portion of the molecule.

[0211] Exemplary CAR Constructs In the CAR example below, the CAR construct is described as "AB domain-hinge-TM domain-CS domain-ICS domain."

[0212] The CAR of the present invention may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any of the following exemplary constructs.

[0213] In one embodiment, a CAR of the invention may be described as h6E6scFvHL-CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 171. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 271.

[0214] In one embodiment, a CAR of the invention may be described as h6E6scFvHL-CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 172. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 272.

[0215] In one embodiment, a CAR of the invention may be described as h6E6scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 173. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 273.

[0216] In one embodiment, a CAR of the invention may be described as h6E6scFvLH-CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 174. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 274.

[0217] In one embodiment, a CAR of the invention may be described as h6E6scFvLH-CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 175. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 275.

[0218] In one embodiment, a CAR of the invention may be described as h6E6scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 176. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 276.

[0219] In one embodiment, a CAR of the invention may be described as h6C10scFvHL-CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 177. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 277.

[0220] In one embodiment, a CAR of the invention may be described as h6C10scFvHL-CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 178. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO:278.

[0221] In one embodiment, a CAR of the invention may be described as h6C10scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 179. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 279.

[0222] In one embodiment, a CAR of the invention may be described as h6C10scFvLH-CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 180. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO: 280.

[0223] In one embodiment, a CAR of the invention may be described as h6C10scFvLH-CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 181. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO:281.

[0224] In one embodiment, a CAR of the invention may be described as h6C10scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence set forth in SEQ ID NO: 182. A nucleic acid sequence encoding such a CAR may comprise the sequence set forth in SEQ ID NO:282.

[0225] Schematic diagrams showing examples of specific CAR constructs of some embodiments are shown in Figures 3A-C.

[0226] In some embodiments, a leader sequence (LS) may be placed upstream of the polynucleotide sequence encoding the aforementioned exemplary CAR. The leader sequence promotes the expression of the CAR on the cell surface. The polynucleotide sequence of such a leader sequence may be as set forth in SEQ ID NO: 260, which encodes the amino acid sequence set forth in SEQ ID NO: 160. Any other sequence that promotes the expression of the CAR on the cell surface may be used.

[0227] A general exemplary schematic of the construct for a LS-containing CAR of the present invention is shown in FIG. 1D.

[0228] In some embodiments, the polynucleotide sequence for expressing the aforementioned exemplary CAR further comprises a sequence encoding a T2A ribosomal skip sequence (also referred to as T2A) and / or a truncated CD19 (also referred to as trCD19). The nucleic acid sequence of T2A can be as provided by SEQ ID NO:269, which encodes the amino acid sequence provided by SEQ ID NO:169. The trCD19 can have the sequence provided by SEQ ID NO:170, which can be encoded, for example, by SEQ ID NO:270.

[0229] A schematic diagram showing such a polynucleotide construct is shown in FIG. 1E.

[0230] When the T2A and trCD19 sequences are placed downstream of the CAR sequence, translation is interrupted by the T2A sequence, resulting in two separate translation products, the CAR protein and the trCD19 protein.

[0231] The present disclosure encompasses nucleic acid sequences encoding any of the CARs disclosed herein.

[0232] Further modifications The CARs of the invention, the nucleotide sequences encoding the CARs of the invention, the vectors encoding the CARs of the invention, and the cells comprising the nucleotide sequences encoding the aforementioned CARs can be further modified, engineered, optimized, or added to provide or select various characteristics. These characteristics can include, but are not limited to, efficacy, persistence, target specificity, reduced immunogenicity, multitargeting, improved immune response, expansion, growth, reduced off-target effects, reduced subject toxicity, improved target cytotoxicity, improved attraction of disease-modifying immune cells, detection, selection, targeting, etc. For example, the cells may be engineered to express another CAR or to have a suicide mechanism, or modified to remove or modify the expression of endogenous receptors or molecules, such as TCR and / or MHC molecules.

[0233] In some embodiments, the vector or nucleic acid sequence encoding the CAR further encodes other genes. The vector or nucleic acid sequence can be constructed to allow for the simultaneous expression of multiple genes using a number of techniques, including co-transfection of two or more plasmids, use of multiple or bidirectional promoters, or generation of bicistronic or multicistronic vectors. Construction of multicistronic vectors can include encoding IRES elements or 2A peptides such as T2A, P2A, E2A, or F2A (see, for example, Kim, JH, et al., "High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice", PLoS One. 2011; 6(4)). In certain embodiments, the nucleic acid sequence or vector encoding the CAR further encodes trCD19 using a T2A ribosomal skip sequence.

[0234] The CAR-expressing cells may further comprise a disruption to one or more endogenous genes. In some embodiments, the endogenous genes encode TCR alpha, TCR beta, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or immune checkpoint proteins such as programmed death-1 (PD-1).

[0235] Effectiveness The CARs of the present invention and cells expressing these CARs can be further modified to improve efficacy against cells expressing target molecules. The cells can be cells expressing ADAM12. The cells expressing ADAM12 can be cancer cells, vascular cells, or any other target disease-related cells. In some embodiments, improved efficacy can be measured by increased cytotoxicity against cells expressing target molecules, for example, cytotoxicity against cancer cells. In some embodiments, improved efficacy can also be measured by increased production of cytotoxic mediators, such as, but not limited to, IFNγ, perforin, and granzyme B. In some embodiments, improved efficacy can be indicated by a reduction in disease signature cytokines, or alleviation of disease symptoms when the CAR-expressing cells are administered to a subject. Other cytokines that can be reduced include TGF-beta, IL-6, IL-4, IL-10, and / or IL-13. Improved efficacy can be indicated by ADAM12-specific immune cell responses, such as T cell cytotoxicity. In the case of cancer, improved efficacy may be indicated by better tumor cytotoxicity, better tumor infiltration, reduced immunosuppressive mediators, reduced weight loss, reduced ascites, reduced tumor burden, and / or extended life span. In the case of autoimmune disease, reduced responsiveness of autoreactive cells or reduced autoreactive T cells, B cells, or Abs may represent improved efficacy. In some embodiments, gene expression profiles may also be investigated to evaluate the efficacy of CAR.

[0236] In one aspect, the CAR-expressing cells are further modified to avoid or neutralize the activity of immunosuppressive mediators, including, but not limited to, prostaglandin E2 (PGE2) and adenosine. In some embodiments, this avoidance or neutralization is direct. In other embodiments, this avoidance or neutralization is mediated through inhibition of protein kinase A (PKA) with one or more binding partners (e.g., ezrin). In certain embodiments, the CAR-expressing cells further express a peptide "regulatory subunit I anchor disruptor" (RIAD). The RIAD is believed to inhibit the association of protein kinase A (PKA) with ezrin, thereby preventing PKA inhibition of TCR activation (Newick K. et al. Cancer Immunol Res. 2016 Jun;4(6):541-51. doi:10.1158 / 2326-6066. CIR-15-0263. Epub 2016 Apr 4).

[0237] In some embodiments, the CAR-expressing cells of the present invention can induce a broad immune response consistent with epitope spreading.

[0238] In some embodiments, the CAR-expressing cells of the present invention further comprise a homing mechanism. For example, the cells may transgenically express one or more stimulatory chemokines, or cytokines or receptors thereof. In certain embodiments, the cells are genetically modified to express one or more stimulatory cytokines. In certain embodiments, one or more homing mechanisms are used to help the cells of the present invention to accumulate more effectively at disease sites. In some embodiments, the CAR-expressing cells are further modified to release inducible cytokines upon CAR activation, for example, to attract or activate innate immune cells to target cells (so-called fourth generation CARs or TRUCKS). In some embodiments, the CARs may co-express homing molecules, for example, CCR4 or CCR2b, to increase transport to disease sites.

[0239] Regulating CAR expression In some cases, it may be advantageous to regulate the activity of CAR or CAR-expressing cells CAR. For example, using a caspase fused to a dimerization domain to induce apoptosis (see, for example, Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18): 1673-1683) can be used as a safety switch in the CAR therapy of the present invention. In another example, CAR-expressing cells can also express an inducible Caspase-9 (iCaspase-9) molecule upon administration of a dimerization drug (e.g., rimiduside (also called AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad))), resulting in activation of Caspase-9 and apoptosis of the cells. The iCaspase-9 molecule contains a chemical inducer of dimerization (CID) binding domain and mediates dimerization in the presence of CID. This causes inducible and selective depletion of CAR-expressing cells. In some cases, iCaspase-9 molecule is encoded by a nucleic acid molecule other than CAR-encoding vector.In some cases, iCaspase-9 molecule is encoded by the same nucleic acid molecule as CAR-encoding vector.iCaspase-9 can provide a safety switch to avoid any toxicity of CAR-expressing cells.See, for example, Song et al.Cancer Gene Ther.2008;15(10):667-75, Clinical Trial Id.No.NCT02107963, and Di et al.N.Engl.J.Med.2011;365:1673-83.

[0240] Alternative strategies for regulating the CAR therapy of the present invention include utilizing small molecules or antibodies that inactivate or turn off CAR activity, for example by deleting CAR-expressing cells, for example by inducing antibody-dependent cell-mediated cytotoxicity (ADCC).For example, the CAR-expressing cells described herein can also express antigens that are recognized by molecules that can induce cell death, for example, ADCC or complement-induced cell death.For example, the CAR-expressing cells described herein can also express receptors that can be targeted by antibodies or antibody fragments. Examples of such receptors include EpCAM, VEGFR, integrins (e.g., integrins αvβ3, α4, αI3 / 4β3, α4β7, α5β1, αvβ3, αv), members of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF receptors, interferon receptors, folate receptors, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a / LFA-1, CD1 5, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5, CD319 / SLAMF7, and EGFR, as well as truncated versions thereof (e.g., versions that preserve one or more extracellular epitopes but lack one or more regions within the cytoplasmic domain). For example, a CAR-expressing cell as described herein may express a truncated epidermal growth factor receptor (EGFR) that lacks signaling capability but retains an epitope recognized by a molecule capable of inducing ADCC, such as cetuximab (ERBITUX®), such that administration of cetuximab induces ADCC and causes subsequent depletion of the CAR-expressing cells (see, e.g., WO 2011 / 056894, and Jonnalagadda et al., "Gene Ther. 2013;20(8)853-860).

[0241] In some embodiments, the CAR cells comprise a polynucleotide encoding a suicide polypeptide, such as, for example, RQR8. See, for example, WO2013 / 15339(1A), which is incorporated herein by reference in its entirety. In CAR cells comprising the polynucleotide, the suicide polypeptide may be expressed on the surface of the CAR cell. The suicide polypeptide may also include a signal peptide at the amino terminus. Another strategy involves expressing a highly compacted marker / suicide gene that combines target epitopes from both the CD32 and CD20 antigens in CAR-expressing cells as described herein that binds to rituximab, resulting in selective depletion of CAR-expressing cells, for example, by ADCC (see, for example, Philip et al., J. Immunol. 1999, 143:1311-1323, 2001). al., "Blood. 2014;124(8)1277-1287). Other methods for depleting CAR-expressing cells described herein include administration of CAMPATH®, a monoclonal anti-CD52 antibody that selectively binds and targets mature lymphocytes, e.g., CAR-expressing cells, for destruction, e.g., by inducing ADCC. In other embodiments, CAR-expressing cells can be selectively targeted using a CAR ligand, e.g., an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can trigger effector cell activity, e.g., ADCC or ADC activity, thereby reducing the number of CAR-expressing cells. In other embodiments, the CAR ligand, e.g., an anti-idiotypic antibody, can bind to an agent (e.g., a toxin) that induces cell death, thereby reducing the number of CAR-expressing cells. Alternatively, the CAR molecule itself can be configured such that its activity can be modulated (e.g., turned on and off), as described below.

[0242] In some embodiments, regulatable CARs (RCARs) that can control CAR activity are desirable to optimize the safety and efficacy of CAR therapy. In some embodiments, RCARs comprise a set of polypeptides, typically two polypeptides in the simplest embodiment, in which the components of standard CARs described herein (e.g., AB domain and ICS domain) are distributed in separate polypeptides or members. In some embodiments, the set of polypeptides comprises a dimerization switch that can bind the polypeptides to each other in the presence of a dimerization molecule, for example, the AB domain to the ICS domain. Further description and exemplary configurations of such regulatable CARs are provided in International Publication No. WO2015 / 090229, which is incorporated herein by reference in its entirety.

[0243] In one aspect, an RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an ICS domain, e.g., a primary ICS domain as described herein, and a first switch domain; 2) an antigen binding member comprising an AB domain that specifically binds to a target molecule as described herein, e.g., as described herein, and a second switch domain. Optionally, the RCAR comprises a TM domain as described herein. In one embodiment, the TM domain may be disposed on the intracellular signaling member, on the antigen binding member, or both. Unless otherwise indicated, when members or elements of an RCAR are described herein, the order may be as provided, but other orders are included as well. In other words, in one embodiment, the order is as described in the document, but in other embodiments, the order may be different. For example, the order of elements on one side of the transmembrane region may be different from the example, e.g., the arrangement of the switch domain relative to the ICS domain may be different, e.g., reversed.

[0244] In some embodiments, the CAR-expressing immune cells may transiently express only the CAR. For example, the cells of the invention may be transduced with an mRNA comprising a nucleic acid sequence encoding the CAR of the invention. In this vein, the invention also includes RNA constructs that may be directly transfected into cells. Methods for generating mRNA for use in transfection involve in vitro transcription (IVT) of a template with specifically designed primers, followed by polyA addition, to generate a construct containing 3' and 5' untranslated sequences ("UTRs"), a 5' cap and / or internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyA tail (typically 50-2000 bases long). The RNA thus produced can efficiently transfect different types of cells. In one embodiment, the template comprises a sequence for the CAR. In one embodiment, the RNA CAR vector is transduced into the cells by electroporation.

[0245] target specificity The CAR-expressing cells of the present invention may further comprise one or more CARs in addition to the first CAR. These additional CARs may or may not be specific to the target molecule of the first CAR. In some embodiments, one or more additional CARs may act as inhibitory or activating CARs. In some aspects, the CARs of some embodiments are stimulatory or activating CARs, and in other aspects, the CARs are costimulatory CARs. In some embodiments, the cells further comprise inhibitory CARs (iCARs, see Fedorov et al., Sci.Transl.Medicine, 2013 Dec;5(215):215ral72), such as CARs that recognize antigens other than the target molecule of the first CAR, and the activation signal delivered through the first CAR is reduced or inhibited by the binding of the inhibitory CAR to its ligand, for example, to reduce off-target effects.

[0246] In some embodiments, the AB domain of the CAR is or is part of an immunoconjugate, and the AB domain is conjugated to one or more heterologous molecules, such as, but not limited to, a cytotoxic agent, an imaging agent, a detectable moiety, a multimerization domain, or other heterologous molecules. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu), chemotherapeutic agents, growth inhibitors, enzymes and fragments thereof, such as nucleases, antibiotics, toxins, such as small molecule toxins or enzymatically active toxins. In some embodiments, the AB domain is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes.

[0247] In some embodiments, to improve persistence, the cells of the invention can be further modified to overexpress pro-survival signals, conversely express anti-survival signals, overexpress Bcl-xL, overexpress hTERT, lack Fas, or express a TGF-β dominant negative receptor. Persistence can also be promoted by administration of cytokines, such as IL-2, IL-7, and IL-15.

[0248] vector The present invention also provides vectors into which a polynucleotide encoding an anti-ADAM12 agent of the present invention is inserted.

[0249] The vector can be, for example, a DNA vector or an RNA vector. The vector can be, for example, but not limited to, a plasmid, a cosmid, or a viral vector. The viral vector can be a DNA virus (may be an adenovirus) vector or an RNA virus (may be a retrovirus) vector. A range of vector types for Ab, antigen-binding Ab fragments, and / or CAR are known in the art (see, for example, Rita Costa A.et al.,Eur J Pharm Biopharm.2010 Feb;74(2):127-38.doi:10.1016 / j.ejpb.2009.10.002.Epub 2009 Oct 22; Frenzel A.et al.Front Immunol.2013;4:217.Published online July 29, 2013 doi:10.3389 / fimmu.2013.00217).

[0250] When the host cell is an insect cell, for example, insect-specific viruses can be used to produce Ab or antigen-binding Ab fragments.Examples of insect-specific viruses include, but are not limited to, the family of Baculoviridae, in particular Autographa californica nuclear polyhedrosis virus (AcNPV).When the host cell is a plant cell, plant-specific viruses and bacteria, such as Agrobacterium tumefaciens, can be used.

[0251] Expression vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, because they allow long-term stable integration of transgenes and their proliferation in daughter cells.Compared to vectors derived from oncoretroviruses such as mouse leukemia virus, lentivirus vectors have an additional advantage in that they can transduce non-proliferating cells such as hepatocytes.These vectors also have the additional advantage of low immunogenicity.This is particularly beneficial for expressing CAR constructs.

[0252] In brief, the expression of the nucleic acid encoding the anti-ADAM12 agent is typically achieved by operably linking the nucleic acid encoding the anti-ADAM12 agent polypeptide or a part thereof to a promoter and incorporating the construct into an expression vector.The vector can be suitable for replication and integration in eukaryotes.A typical cloning vector includes transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of desired nucleic acid sequences.

[0253] The expression construct of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocol.Gene delivery method is known in the art.See, for example, U.S. Patent No. 5,399,346, U.S. Patent No. 5,580,859, U.S. Patent No. 5,589,466, which are incorporated herein by reference in their entirety.In another embodiment, the present invention provides a gene therapy vector.

[0254] Nucleic acids can be cloned into several 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.

[0255] Furthermore, expression vectors can be provided to cells in the form of viral vectors.Viral vector technology is 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, gamma-retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0256] Several 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 retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0257] 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 some promoters have recently been shown to contain functional elements downstream of the start site as well. In many cases, the spacing between promoter elements is flexible, so that promoter function is maintained even if elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to as much as 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can function cooperatively or independently to activate transcription.

[0258] A variety of promoter sequences may be used, including, but not limited to, the immediate early cytomegalovirus (CMV) promoter, the CMV-actin-globin hybrid (CAG) promoter, the elongation growth factor-1α (EF-1α), the Simian Virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the long terminal repeat (LTR) promoter of the human immunodeficiency virus (HIV), the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters (e.g., but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter). Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is desired, or turn off the 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.

[0259] To evaluate the expression of the CAR polypeptide or a part thereof, the expression vector introduced into the cell can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the collection of cells that are to be transfected or infected by the viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be adjacent to the appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0260] In some embodiments, the selectable marker gene comprises a nucleic acid sequence encoding truncated CD19 (trCD19). When a marker such as trCD19 that can be expressed on the cell surface is used, the expression of the marker can be determined through any available technique, including but not limited to flow cytometry or immunofluorescence assay. The expression of such a marker typically indicates successful introduction and expression of the transgene introduced with the marker gene. Thus, the cell expressing the anti-ADAM12 agent of the present invention can be selected based on, for example, the expression of the marker.

[0261] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that code for a polypeptide whose expression is expressed by some easily detectable property, e.g., enzymatic activity. The expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes that code for luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are known and may be prepared using known techniques or may be commercially obtained. In general, the construct with the minimal 5' flanking region that shows the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate drugs for their ability to modulate transcription caused by the promoter.

[0262] Transfection / transduction 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 transfected into host cells by physical, chemical or biological means.

[0263] A flow chart showing potential methods for producing isolated CAR-expressing cells for transduction of a CAR construct to obtain a CAR-expressing cell is provided in FIG. 4.

[0264] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0265] Biological methods for introducing a polynucleotide of interest into host cells include the use of DNA vectors and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, for example human, cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0266] Chemical means for introducing polynucleotides into host cells include macromolecular complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid 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).

[0267] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acid into a host (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained in a micelle, or complexed with a micelle, 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 may have a "collapsed" structure. They may also simply be dispersed in the solution, and may form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0268] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo., dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of single and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before a closed structure is formed, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., "1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from the usual vesicular structure are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0269] Regardless of the method used to introduce exogenous nucleic acid into a host cell or otherwise expose the cell to the inhibitor of the present invention, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biological" assays known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of a particular peptide by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0270] cell Also provided are cells, cell aggregates, and cell-containing compositions, such as cells that contain the nucleic acid sequence encoding the anti-ADAM12 agent of the present invention.Cells expressing anti-ADAM12 Ab or antigen-binding Ab fragments can be used to harvest Ab or antigen-binding Ab fragments.Cells expressing anti-ADAM12 CAR can be administered to subjects or incorporated into compositions that are administered to subjects.Compositions include pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.

[0271] Also provided are therapeutic methods for administering the Abs or Ab fragments, or cells and compositions to a subject, eg, a patient.

[0272] cell type Accordingly, cells expressing an anti-ADAM12 agent of the invention are also provided.

[0273] Any suitable cell can be used to express anti-ADAM12 Ab or antigen-binding Ab fragment.For example, cell can be (i) prokaryotic cell such as gram-negative bacteria and gram-positive bacteria, or (ii) eukaryotic cell such as yeast, filamentous fungus, protozoan, insect cell, plant cell and mammalian cell (reviewed in Frenzel A.et al.Front Immunol.2013;4:217.Published online on July 29, 2013 doi:10.3389 / fimmu.2013.00217).

[0274] Specific examples of Gram-negative bacteria suitable for producing Abs or antigen-binding Ab fragments include, but are not limited to, E. coli, Proteus mirabilis, and Pseudomonas putidas. Specific examples of Gram-positive bacteria include, but are not limited to, Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lacto-bacillus zeae / casei, and Lactobacillus paracasei. Specific examples of yeast suitable for producing Abs or antigen-binding Ab fragments include, but are not limited to, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, and Yarrowia lipolytica. Specific examples of filamentous fungi suitable for producing Abs or antigen-binding Ab fragments include, but are not limited to, Trichoderma and Aspergillus genera, A. niger (subgenus A. awamori), Aspergillus oryzae, and Chrysosporium lucknowense. Specific examples of protozoa suitable for producing Abs or antigen-binding Ab fragments include, but are not limited to, Leishmania tarentolae. Specific examples of insect cells suitable for producing Abs or antigen-binding Ab fragments include, but are not limited to, insect cell lines such as Spodoptera frugiperda Sf-9 and Sf-21, Drosophila melanogaster DS2 cells, Trichopulsia ni High Five cells (BTI-TN-5B1-4), or D. melanogaster Schneider2 (S2) cells. These cells can be efficiently transfected with insect-specific viruses from the family Baculoviridae, in particular Autographa californica nuclear polyhedrosis virus (AcNPV).Specific examples of mammalian cells suitable for production of Abs or antigen-binding Ab fragments include, but are not limited to, Chinese hamster ovary (CHO) cells, the human embryonic retina cell line Per.C6 [Crucell, Leiden, Netherlands], CHO-derived cell lines such as K1-, DukXB11-, Lec13, and DG44-cell lines, mouse myeloma cell lines such as SP 2 / 0, YB 2 / 0, and NS0 cells, GS-NSO, hybridoma cells, baby hamster kidney (BHK) cells, and the human embryonic kidney cell lines HEK293, HEK293T, HEK293E, and the human neural progenitor cell line AGE1.HN (Probiogen, Berlin, Germany).

[0275] Alternatively, genetically modified organisms such as transgenic plants and animals may be used. Exemplary plants that may be used include, but are not limited to, tobacco, corn, duckweed, Chlamydomonas reinhardtii, Nicotiana tabacum, Nicotiana benthamiana, and Nicotiana benthamiana. Exemplary animals that may be used include, but are not limited to, mice, rats, and chickens.

[0276] To express an anti-ADAM12 CAR, the cell is generally a eukaryotic cell, such as a mammalian cell, typically a human cell, more typically a primary human cell, e.g., an allogeneic or autologous donor cell. The cell for introducing the CAR can be isolated from a sample, e.g., a biological sample, such as a sample obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is a subject with a disease or condition, or a subject in need of cell therapy, or a subject to whom cell therapy is to be performed. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, from which the cell is isolated, processed, and / or manipulated. In some embodiments, the cell is derived from blood, bone marrow, lymph, or lymphoid organs and is a cell of the immune system, e.g., a cell of innate or adaptive immunity, e.g., a bone marrow cell, including a monocyte, macrophage, dendritic cell, neutrophil, eosinophil, basophil, or adipocyte, or a lymphocyte, typically including a T cell and / or a NK cell. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as cells isolated directly from a subject and / or cells isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation, e.g., total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof.

[0277] Alternatively, immortalized cells or cell lines can be used to express the CAR of the present disclosure. Such examples include, but are not limited to, T cell lines, CD4+ T cell lines, CD8+ T cell lines, regulatory T cell lines, NK-T cell lines, NK cell lines (e.g., NK-92), monocyte lines, macrophage lines, dendritic cell lines, and mast cell lines. Furthermore, the desired cell type for CAR expression, for example, T cells or NK cells, can be generated from stem cells, such as embryonic stem cells, iPSCs, or hematopoietic stem cells.

[0278] For subjects treated with cells expressing anti-ADAM12 CAR, the cells may be allogeneic and / or autologous. Some of the methods include off-the-shelf methods. In some aspects, such as off-the-shelf techniques, the cells are pluripotent and / or multipotent, e.g., stem cells, e.g., induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject, preparing, processing, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0279] In some embodiments, the cell is a T cell. Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and subtypes thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring adaptive regulatory T (TREG) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0280] In some embodiments, the cells are natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TIL), lymphokine-activated killer (LAK) cells, etc. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. The expressing CAR-expressing phagocytes may be capable of binding to and phagocytosis or engulfing target cells (Morrissey MA et al., Elife. 2018 Jun 4; 7. pii: e36688. doi: 10.7554 / eLife.36688).

[0281] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, e.g., mouse, rat, non-human primate, and pig.

[0282] Cell acquisition for anti-ADAM12 CAR expression For cells to express anti-ADAM12 CAR, the source of cells can be obtained from a subject through various non-limiting methods prior to expansion and genetic modification. Cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and disease sites such as fibrotic sites or tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art may be used. In some embodiments, cells can be derived from healthy donors, patients diagnosed with cancer, or patients diagnosed with infectious diseases. In some embodiments, cells can be part of a mixed population of cells that exhibit different phenotypic characteristics.

[0283] Thus, the cells in some embodiments are primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from subjects, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction by viral vectors), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or samples that are processed. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, and include processed samples derived therefrom.

[0284] In some embodiments, the sample from which the cells are derived or from which the cells are isolated is blood or blood-derived sample, or is or is derived from Cresis or leukapheresis therapy product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, fibrotic tissue, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom.Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0285] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some embodiments, contains cells other than red blood cells and platelets.

[0286] Also provided herein is a cell line obtained from a cell transformed according to any of the above-mentioned methods.Also provided herein is a modified cell that is resistant to immunosuppressive treatment.In some embodiments, the isolated cell of the present invention comprises a polynucleotide encoding a CAR.

[0287] cell purification In some embodiments, the isolation of cells includes one or more preparative and / or affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove undesirable components, enrich for desired components, lyse or remove cells that are sensitive to a particular reagent. In some examples, cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0288] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing solution is free of calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing, e.g., Ca++ / Mg++ free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0289] In some embodiments, the isolation method involves the separation of different cell types based on the expression or presence in the cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. This may be particularly useful for isolating CAR-expressing cells. In certain embodiments, the surface maker is trCD19. In some embodiments, any known method for separating based on such markers may be used. In some embodiments, the separation is an affinity or immunoaffinity based separation. For example, in some aspects, the isolation involves the separation of cells and cell aggregates based on the cellular expression or expression level of one or more markers, typically cell surface markers, e.g., by incubation with an antibody or binding partner that specifically binds to such markers, typically followed by a washing step and separation of the cells bound to the antibody or binding partner from those cells that are not bound to the antibody or binding partner.

[0290] Such a separation step can be based on positive selection, which retains cells that bind to the reagent for further use, and / or negative selection, which retains cells that do not bind to the antibody or binding partner. In some instances, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful when antibodies that specifically distinguish cell types in a heterogeneous population are not available, and as a result separation is best performed based on markers expressed by cells outside the desired population.

[0291] In some embodiments, multiple separation steps are performed, with the positively or negatively selected fractions from one step being subjected to another separation step (e.g., subsequent positive or negative separation). In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, for example, by incubating cells with multiple antibodies or binding partners, each specific for a marker targeted by negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on different cell types.

[0292] For example, in some embodiments, specific subsets of T cells, e.g., positive cells or cells expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. For example, CD3+ T cells can be positively selected using CD3-conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0293] In some embodiments, isolation is performed by enrichment of specific cell populations by positive selection, or depletion of specific cell populations by negative selection. In some embodiments, positive or negative selection is achieved by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed or expressed at high levels (marker high) relative to the positively or negatively selected cells (marker +), respectively.

[0294] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells such as B cells, monocytes, or other white blood cells such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection of markers expressed or expressed to a relatively high degree on one or more subsets of naive, memory, and / or effector T cells.

[0295] In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subset. In some embodiments, enrichment of central memory T (TCM) cells is performed to increase efficacy, such as improving long-term survival, expansion, and / or engraftment after administration, and in some aspects is particularly robust in such subsets. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, TCM-enriched CD8+ T cells and CD4+ T cells are combined to further enhance efficacy. In several embodiments, memory T cells are present in both the CD62L+ and CD62L subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted of the CD62L-CD8+ and / or CD62L+CD8 fractions using, for example, anti-CD8 and anti-CD62L antibodies.

[0296] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127. In some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells begins with a negative fraction of cells selected on the basis of CD4 expression, which undergoes negative selection based on expression of CD14 and CD45RA, followed by positive selection on the basis of CD62L. In some aspects, such selections are performed simultaneously, while in other aspects, they are performed sequentially in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, and both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally following one or more further positive or negative selection steps.

[0297] In some embodiments, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, e.g., a paramagnetic bead (e.g., Dynalbead or MACS beads). The magnetically responsive material, e.g., a particle, generally connects directly or indirectly to a molecule, e.g., a binding partner (e.g., an antibody) that specifically binds to a surface marker present on the cell, cells, or collection of cells that it is desired to separate (e.g., negatively or positively select).

[0298] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. Many known magnetically responsive materials have been used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and European Patent Specification No. EP 452342 B, which are incorporated herein by reference. Colloidal-sized particles, such as those described in Owen, U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084, are other examples.

[0299] Incubation is generally carried out under conditions where the antibody or binding partner, or a molecule that specifically binds to such an antibody or binding partner and that is bound to the magnetic particle or bead (e.g., a secondary antibody or other reagent), specifically binds to a cell surface molecule if present on cells in the sample.

[0300] In some embodiments, the sample is placed in a magnetic field, and those cells with magnetically responsive or magnetizable particles attached thereto are attracted to the magnet and separated from unlabeled cells. In positive selection, the cells that are attracted to the magnet are retained, and in negative selection, the cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, and the positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0301] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are connected to cells via coating with a primary antibody specific for one or more markers. In certain embodiments, cells are labeled with a primary antibody or binding partner, rather than beads, and then cell type-specific secondary antibody or other binding partner (e.g., streptavidin)-coated magnetic particles are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0302] In some embodiments, the magnetically responsive particles remain attached to the cells which are subsequently incubated, cultured, and / or manipulated, and in some aspects, the particles remain attached to the cells for administration to the patient. In some embodiments, the magnetizable particles or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known, including, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0303] In certain embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user handling, and / or contamination. In one example, the system is a system described in International Patent Application Publication No. WO2009 / 072003, or US2011 / 0003380(A1).

[0304] In some embodiments, the system or device performs one or more, e.g., all, of the isolation, processing, manipulation, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or a computer program in communication with the system or device, which allows a user to program, control, evaluate the results, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0305] In some embodiments, the cell aggregates described herein are collected and enriched (or depleted) via flow cytometry, where cells stained for multiple cell surface markers are retained in the fluid stream. In some embodiments, the cell aggregates described herein are collected and enriched (or depleted) via preparative-scale (FACS) sorting. In certain embodiments, the cell aggregates described herein are collected and enriched (or depleted) by using a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140; Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In either case, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets with high purity.

[0306] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, cell separation based on binding of antibodies or other binding partners specific to one or more cell surface markers is performed in a fluid flow, such as fluorescence activated cell sorting (FACS), including preparative scale (FACS) and / or microelectromechanical system (MEMS) chips, in combination with a flow cytometry detection system. Such methods allow positive and negative selection based on multiple markers simultaneously.

[0307] In some embodiments, the methods involve preparation of white blood cells from peripheral blood by lysing red blood cells and density-based cell separation methods, such as centrifugation through a Percoll or Ficoll gradient.

[0308] In any of the above separation steps, separation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.For example, positive selection or enrichment of a particular type of cell, e.g., cells expressing a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker.Similarly, negative selection, removal, or depletion of a particular type of cell, e.g., cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0309] Cell preparation and expansion In some embodiments, the methods provided include culturing, incubation, culturing, and / or genetic manipulation steps. For example, in some embodiments, methods are provided for incubating and / or manipulating the depleted cell aggregates and culture starting compositions.

[0310] Thus, in some embodiments, the cell aggregates are incubated in a culture starting composition. The incubation and / or manipulation may be performed in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other vessel for culturing or growing cells.

[0311] In some embodiments, the cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. The incubation step may include culturing, culturing, stimulating, activating, and / or expanding.

[0312] In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of stimulatory agents. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells within the aggregate, mimic antigen exposure, and / or prime the cells for genetic engineering, such as the introduction of recombinant antigen receptors. The cells of the invention may be cultured under conditions described in, for example, but not limited to, 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, 7,024,576, 7,102,577, 7,200,578, 7,300,579, 7,400,579, 7,500,579, 7,500,579, 7,600,579, 7,700,579, 7,800,579, 7,900,579, 7,102,579, 7,200,579, 7,300,579, 7,400,579, 7,500,579, 7,500,579, 7,600,579, 7,7 ... The cells may be activated and expanded using methods generally described in U.S. Patent Application Publication Nos. 67,318, 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. 2006 / 0121005. Conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimuli such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0313] In particular, with respect to CAR-expressing cells, T cells can be expanded in vitro or in vivo.Generally, the T cells of the present invention can be expanded, for example, by contact with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the T cells to generate an activation signal for the T cells.For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate an activation signal for the T cells.

[0314] In some embodiments, T cell populations can be stimulated in vitro, for example, by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. In some embodiments, T cell populations can be stimulated in vitro by contacting with muromonab-CD3 (OKT3). Co-stimulation of accessory molecules on the surface of T cells uses a ligand that binds to the accessory molecule. For example, T cell populations can be contacted with anti-CD3 antibody and anti-CD28 antibody under conditions appropriate to stimulate proliferation of T cells. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Media 1640® or X-vivo 5®, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, IL-21, TGF-β, and TNF, or any other additives for cell growth known to those of skill in the art. In a preferred embodiment, T cells are stimulated in vitro by exposure to OKT3 and IL-2. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640®, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1®, and X-Vivo 20®, Optimizer, and are supplemented with amino acids, sodium pyruvate, and vitamins, and are either serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells that will be infused into subjects.Target cells are maintained under conditions necessary to support growth, e.g., appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2). T cells exposed to various stimulation times may exhibit different characteristics.

[0315] In some embodiments, the isolated cells of the present invention can be expanded by co-culturing with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0316] In some embodiments, when cells are expanded in vivo, at least one cell of the present invention may be administered to a subject, and administration may cause cell expansion in the subject, resulting in cell aggregation. Alternatively, the nucleic acid sequence or vector of the present invention may be administered to a subject. When the nucleic acid sequence or vector is taken up by cells in the subject, and the cells grow or expand in the subject, cell aggregation of the present invention may occur in the subject.

[0317] In certain embodiments, the resulting population of cells is maintained in the subject for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 2 years, or at least 3 years following administration.

[0318] In some embodiments, the T cells are expanded by adding to the culture initiation composition feeder cells (e.g., non-dividing peripheral blood mononuclear cells (PBMCs)) (e.g., such that the resulting collection of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial collection to be expanded) and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000-3600 rad to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the T cell collection.

[0319] In some embodiments, the preparation method includes a step for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and / or manipulation. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and to some extent monocytes in the cell aggregates. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters may be used in some aspects. One example includes using PBS with 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. This is then diluted 1:1 with medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80°C at a rate of 1 degree per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0320] Isolation of Ab or antigen-binding Ab fragments from cell cultures Cells (e.g., hybridomas) producing the Abs or antigen-binding Ab fragments of the invention can be grown in vivo in a culture medium suitable for this purpose (e.g., D-MEM or RPMI-1640), or as ascites, using standard methods. Abs or antigen-binding Ab fragments secreted by the cells can be isolated from the culture medium, ascites fluid, or serum using conventional immunoglobulin purification procedures, such as, for example, but not limited to, protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography (Ma H. et al., Methods. 2017 Mar 1;116:23-33. doi:10.1016 / j.ymeth.2016.11.008. Epub 2016 Nov 18; Shukla AA et al. Trends Biotechnol. 2010 May;28(5):253-61. doi:10.1016 / j.tibtech.2010.02.001. Epub 2010 Mar 19; Arora S. et al., Methods. 2017 Mar 1;116:84-94.doi:10.1016 / j.ymeth.2016.12.010.Epub 2016 Dec 22).

[0321] Methods for expressing, isolating, and evaluating multispecific and bispecific Abs and antigen-binding Ab fragments are also known in the art (see, e.g., Brinkmann U. et al., MAbs. 2017 Feb-Mar;9(2):182-212. Prepublished online Jan. 10, 2017, doi:10.1080 / 19420862.2016.1268307; Dimasi N. et al. Methods. 2018 Aug 11. pii:S1046-2023(18)30149-X. doi:10.1016 / j.ymeth.2018.08.004).

[0322] Therapeutic Applications The anti-ADAM12 agents of the present invention (Abs, antigen-binding Ab fragments, multispecific Abs, multispecific antigen-binding Ab fragments, ADCs that bind ADAM12, or CARs), nucleic acids encoding such agents, vectors encoding such agents, isolated cells obtained by the above-mentioned methods, or cell lines derived from such isolated cells, and / or pharmaceutical compositions comprising them can be used as medicaments in the treatment of diseases, disorders, or conditions in a subject. In some embodiments, such medicaments can be used to treat ADAM12-related diseases or conditions.

[0323] Target Diseases and Conditions The ADAM12-associated condition can be, for example, but is not limited to, cancer, fibrosis, autoimmunity, cardiovascular condition, allergic condition, respiratory disease, nephropathy, neurological disease, muscular disease, liver disease, metabolic syndrome, infection, or an inflammatory disorder.

[0324] In certain embodiments, the anti-ADAM12 agents of the invention may be used to treat cancer. ADAM12 is upregulated and / or plays a pathological role in a wide variety of cancers, including, but not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumors, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, and thyroid cancer (Kveiborg M.et al.,Int J Biochem Cell Biol.2008;40(9):1685-702.doi:10.1016 / j.biocel.2008.01.025.Epub 2008 Feb 1; Le Pabic H.et l.,Hepatology.2003 May;37(5):1056-66, Skubitz KMet al.,J Lab Clin Med.2004 Feb;143(2):89-98., Carl-McGrath S.et al.,Int J Oncol.2005 Jan;26(1):17-24, Mochizuki S.et al.,Cancer Sci.2007 May;98(5):621-8.Epub 2007 Mar 9, Colombo C.et al.,J Pathol.2011 Dec;225(4):574-82. doi:10.1002 / path.2951.Epub 2011 Aug 8, Sookprasert A.et al.,Asian Pac J Cancer Prev.2012;13 Suppl:3-6, Uehara E. et al., Int J Oncol.2012 May;40(5):1414-22.doi:10.3892 / ijo.2012.1339.Epub 2012 Jan 20, Baren JPet al.,Br J Cancer.2012 Jun 26;107(1):143-9.doi:10.1038 / bjc.2012.239.Epub 2012 Jun 7, Rao VHet al.,Oncogene.2012 Jun 7;31(23):2888-98.doi:10.1038 / onc.2011.460.Epub 2011 Oct 10;Kanakis D. et al.,Dis Markers.2013;34(2):81-91.doi:10.3233 / DMA-120953、Georges S.et al.,Eur J Cancer.2013 Jun;49(9):2253-63.doi:10.1016 / j.ejca.2013.02.020.Epub 2013 Mar 13、Cireap N.et al.,Pathol Oncol Res.2013 Oct;19(4):755-62.doi:10.1007 / s12253-013-9639-8.Epub 2013 May 6、Bilgin Dogru E.et al.,Tumour Biol.2014 Nov;35(11):11647-53.doi:10.1007 / s13277-014-2514-8.Epub 2014 Aug 20、Cheon D.J.Et al.,Carcinogenesis.2015 Jul;36(7):739-47.doi:10.1093 / carcin / bgv059.Epub 2015 Apr 29、Rao V.H.et al.,Mol Carcinog.2015 Oct;54(10):1026-36.doi:10.1002 / mc.22171.Epub 2014 May 5、Li Z.et al.,Oncol Rep.2015 Dec;34(6):3231-7、Liu G.et al.,Oncol Rep.2016 Nov;36(5):3005-3013.doi:10.3892 / or.2016.5064.Epub 2016 Sep 5、Frohlich C.et al.,Clin Cancer Res.2006 Dec 15;12(24):7359-68、Roy R.et al.,Mol Cancer Res.2017 Nov;15(11):1608-1622.doi:10.1158 / 1541-7786.MCR-17-0188.Epub 2017 Aug 1、Xiong L.et al.,J Proteomics.2018 Jun 30;182:34-44.doi:10.1016 / j.jprot.2018.04.033.Epub 2018 May 2、Veenstra V.L.et al.,Oncogenesis.2018 Nov 16;7(11):87.doi:10.1038 / s41389-018-0096-9). The anti-ADAM12 agent of the present invention may be used to treat any of the above-mentioned cancers. Upregulation is particularly high in breast cancer, and interestingly, ADAM12 is known to induce overexpression of HER2 / neu, a known breast cancer antigen (Nyren-Erickson EKBiochim Biophys Acta.2013 Oct;1830(10):4445-55.doi:10.1016 / j.bbagen.2013.05.011.Epub 2013 May 13). Therefore, breast cancer is one of the preferred target diseases of the present invention. The anti-ADAM12 agent of the present invention may also be used to treat any other cancer in which ADAM12 is upregulated or has a pathological role. .

[0325] In certain embodiments, the anti-ADAM12 agents of the invention may be used to treat a non-cancer disease or condition. ADAM12 is upregulated and / or plays a pathological role in many other diseases and conditions, including, but not limited to, Alzheimer's disease, osteoarthritis, muscular dystrophy, multiple sclerosis, fibrosis, cardiac hypertrophy, dermal fibrosis and interstitial lung disease in systemic sclerosis, renal fibrosis, peripheral arterial disease, endometriosis, and Dupuytren's disease (Kveiborg M.et al.,Int J Biochem Cell Biol.2008;40(9):1685-702. doi:10.1016 / j.biocel.2008.01.025.Epub 2008 Feb 1; Harold D.et al.,Am J Med Genet B Neuropsychiatr Genet.2007 Jun 5;144B(4):448-52; Kerna I.et al.,Rheumatol Int.2012 Feb;32(2):519-23.doi:10.1007 / s00296-010-1717-6.Epub 2011 Jan 22, Dulauroy S.et al.,Nat Med.2012 Aug;18(8):1262-70.doi:10.1038 / nm.2848.Epub 2012 Jul 29, Berry E.et al.,J Vasc Res.2013;50(1):52-68.doi:10.1159 / 000345240.Epub 2012 Nov 17, Taniguchi T.et al.,J Eur Acad Dermatol Venereol.2013 Jun;27(6):747-53.doi:10.1111 / j.1468-3083.2012.04558.x.Epub 2012 Apr 28, Ramdas V.et al.,Am J Pathol.2013 Dec;183(6):1885-1896.doi:10.1016 / j.ajpath.2013.08.027.Epub 2013 Oct 6, Dokun AOet al.,Am J Physiol Heart Circ Physiol.2015 Sep;309(5):H790-803.doi:10.1152 / ajpheart.00803.2014.Epub 2015 Jul 10, Miller MAet al.,Sci Rep.2015 Oct 19;5:15150.doi:10.1038 / srep15150; Sedic M. et al.,(2012) Using Functional Genomics to Identify Drug Targets:A Dupuytren's Disease Example.In:Larson R. (ed.) Bioinformatics and Drug Discovery.Methods in Molecular Biology(Methods and Protocols), vol 910.Humana Press, Totowa, NJ). Thus, in some embodiments, the anti-ADAM12 agents and compositions of the present disclosure may be used to treat Alzheimer's disease, osteoarthritis, muscular dystrophy, multiple sclerosis, fibrosis, cardiac hypertrophy, skin fibrosis and interstitial lung disease in systemic sclerosis, renal fibrosis, peripheral arterial disease, endometriosis, or Dupuytren's disease. In some embodiments, the anti-ADAM12 agents and compositions of the present disclosure may be used to treat any condition characterized by increased ADAM12 expression.

[0326] subject The subject referred to herein may be any living subject. In a preferred embodiment, the subject is a mammal. The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, e.g., mice and hamsters, and mammals of the order Logomorpha, e.g., rabbits. The mammal may be a mammal from the order Carnivora, including Feline (cat) and Canine (dog). The mammal may be a mammal from the order Artiodactyla, including Bovine (cow) and Swine (pig), or Perssodactyla, including Equine (horse). The mammal may be a mammal from the order Primate, Ceboid, or Simoid (monkey), or the order Anthropoid (human and ape).

[0327] In some embodiments, the subject to which the Ab, antigen-binding Ab fragment, ADC, CAR-expressing cell, cell, cell aggregate, or composition is administered is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject may be male or female and may be of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some examples, the patient or subject is a validated animal model for evaluating disease, adoptive cell therapy, and / or toxicity outcomes, such as cytokine release syndrome (CRS).

[0328] In some embodiments, the subject has persistent or recurrent disease, for example, after treatment with another immunotherapy and / or other therapy. In some embodiments, this administration effectively treats the subject even though the subject has become resistant to another therapy. In some embodiments, the subject has not relapsed but is determined to be at risk of relapse, such as at high risk of relapse, and thus the compound or composition is administered prophylactically, for example, to reduce or prevent the likelihood of relapse.

[0329] In some embodiments, the method includes administration of Ab, Ab fragment, ADC, or CAR-expressing cell, or composition comprising such anti-ADAM12 agent to a subject, tissue, or cell, such as one having, at risk for, or suspected of having an ADAM12-related disease, condition, or disorder, cancer, fibrosis, autoimmunity, cardiovascular condition, allergic condition, respiratory disease, nephropathy, neurological disease, muscular disease, liver disease, metabolic syndrome, infection, and inflammatory disorder. In some embodiments, the anti-ADAM agent and / or composition is administered to a subject having the particular disease or condition to be treated, for example, via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the anti-ADAM12 agent or composition is administered to a subject, such as a subject having or at risk for a disease or condition. In some aspects, the method thereby treats, e.g., ameliorates, one or more symptoms of a disease or condition, for example, by reducing, inhibiting, or inactivating ADAM12 and / or ADAM12-expressing cells.

[0330] cell origin For methods of anti-ADAM12 CAR therapy in which a host cell or cell population is administered, the cells may be xenogeneic, allogeneic, or autologous to the subject.

[0331] In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is performed by autologous transfer, where cells are isolated and / or otherwise prepared from the subject receiving cell therapy, or a sample derived from such a subject.Thus, in some aspects, cells are derived from a subject, e.g., a patient, in need of treatment, and the cells are administered to the same subject after isolation and processing.

[0332] In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is performed by allogeneic transfer, in which cells are isolated and / or otherwise prepared from a subject (e.g., a first subject) different from the subject receiving cell therapy or the subject who will ultimately receive cell therapy. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first subject and the second subject are genetically identical. In some embodiments, the first subject and the second subject are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0333] In certain embodiments, if the cell is a T cell and is not autologous to the subject, the expression of the cell's endogenous T cell receptor (TCR) may be suppressed or hindered. TCR expression may be suppressed through any suitable technique, for example, by silencing any compartment of the endogenous TCR using tools such as, but not limited to, siRNA, shRNA, microRNA, or artificial microRNA. Alternatively, the TCR gene may be disrupted or deleted through any suitable technique, for example, using the CRISPR / Cas system, transcription activator-like effector nuclease (e.g., TALEN®), or zinc finger nuclease (ZFN). Suppression or disruption of the TCR may reduce or prevent the undesirable effect that the TCR recognizes antigens in the subject as foreign and causes an immune response against the subject, an immune attack often referred to as graft-versus-host disease (GVHD).

[0334] In certain embodiments, when the cells (donor cells) are not autologous to the subject, the expression of endogenous MHC or HLA genes can be suppressed or destroyed, which can be achieved through any suitable technique, such as, but not limited to, siRNA, shRNA, microRNA, artificial microRNA, or gene editing using CRISPR / Cas systems, transcription activator-like effector nucleases (e.g., TALEN®), or zinc finger nucleases (ZFNs). Suppression or destruction of MHC or HLA genes can reduce or prevent the undesirable effect that the subject's endogenous T cells recognize the antigens of the donor cells presented on the MHC molecules of the donor cells as foreign, triggering an immune response against the donor cells and increasing the persistence of the administered cells in the subject. Cells expressing anti-ADAM12 Ab or antigen-binding Ab fragments, or compositions comprising them, can also be administered to the subject. In some embodiments, B cells or plasma cells expressing anti-ADAM12 Ab or antigen-binding Ab fragments can be adoptively transferred.

[0335] functional activity In one embodiment, the present invention includes a type of cell therapy in which isolated cells are genetically modified to express a CAR against ADAM12, and the CAR cells are injected into a subject in need thereof. Such administration can promote the activation of cells (e.g., T cell activation) in a target molecule-specific manner, so that disease or disorder cells are targeted for destruction. If the cell is a T cell, the cell, such as a CAR T cell, can replicate in vivo, resulting in long-term persistence that can result in sustained control of disease, disorder, or condition associated with ADAM12, cancer, fibrotic condition, cardiovascular condition, inflammatory condition, or autoimmune condition.

[0336] In one embodiment, the isolated cells of the present invention can undergo in vivo expansion and persist for a long period of time. In another embodiment, when the isolated cells are T cells, the isolated T cells of the present invention evolve into specific memory T cells that can be reactivated to inhibit the growth of any additional target molecule-expressing cells. Upon encounter, T cells can differentiate into a central memory-like state in vivo and then eliminate target cells that express surrogate antigens. Similarly, in certain embodiments where the isolated cells are B cells, the isolated B cells can evolve into memory B cells that can be reactivated to inhibit the growth of any additional target molecule-expressing cells.

[0337] Without wishing to be bound by any particular theory, the immune response induced by the isolated anti-ADAM12 agent modified immune cells can be an active or passive immune response.In addition, the immune response mediated by the anti-ADAM12 agent can be part of an adoptive immunotherapy approach, where the anti-ADAM12 agent modified immune cells induce the anti-ADAM12 agent AB domain specific immune response.

[0338] In certain embodiments, anti-ADAM12 agent expressing cells are modified in any number of ways to increase their therapeutic or prophylactic efficacy.For example, anti-ADAM12 agent can be directly or indirectly conjugated to targeting moiety via linker.The practice of conjugating compound (e.g., CAR) to targeting moiety is known in the art.See, for example, Wadwa et al., J.Drug Targeting 3:1 1 1 (1995) and U.S. Patent No. 5,087,616.

[0339] Once the cells are administered to a subject (e.g., a human), the biological activity of the engineered cell aggregates and / or antibodies in some embodiments is measured by any of several known methods. Parameters to be evaluated include specific binding of engineered T cells or natural T cells or other immune cells to antigens in vivo (e.g., by imaging) or ex vivo (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of engineered cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assay described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009), and Herman et al. J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of cells can also be measured by assaying the expression and / or secretion of certain mediators, such as GM-CSF, IL-6, RANTES (CCL5), TNF-α, IL-4, IL-10, IL-13, IFN-γ, granzyme B, perforin, CD107a, or IL-2.

[0340] In some aspects, biological activity is measured by evaluating clinical outcomes such as reduction in disease symptoms. In the case of autoimmune disease, reduction in autoreactive T cells, B cells, or Abs, as well as reduction in inflammation, may represent successful biological activity. In the case of cancer, improved efficacy may be indicated by better infiltration of disease-resolving immune cells into tumors, reduction in tumor size, or reduction in ascites. In some embodiments, gene expression profiles may also be investigated to assess activity.

[0341] target cell The cells that can be targeted by any anti-ADAM12 agent of the present invention include any ADAM12-expressing cells.Target cells can be present in any part of the subject's body, including blood or lymph circulation and disease-affected tissue.For example, when target disease is solid cancer, disease-affected tissue includes, but is not limited to, bladder, bone, brain, breast, colon, rectum, connective tissue, esophagus, dermis, subcutaneous connective tissue, neuron, squamous cell, liver, lung, epidermis, esophagus, stomach, oligodendroglioma, oral tissue, oral squamous cell, ovary, pancreas, prostate, skin, lung, and thyroid.Alternatively, target cells can be blood cells or hematopoietic cells.

[0342] Preferably, the anti-ADAM12 agent-expressing cells of the present invention are used to treat cancers in which ADAM12 is upregulated. In particular, the cells of the present invention can be used to treat breast cancer, lung cancer, brain cancer, stomach cancer or skin cancer.

[0343] Generally, cells positive for ADAM12 can be identified by known methods, such as immunofluorescence or flow cytometry using specific antibodies, or alternatively, by cytotoxicity against target cells.The method of testing anti-ADAM12 agents for their ability to recognize target cells and antigen specificity is known in the art.For example, Clay et al., J.Immunol., 163:507-513 (1999) teaches a method of measuring the release of cytokines (e.g., interferon gamma, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), or interleukin 2 (IL-2)).In addition, CAR function can be evaluated by measuring the cytotoxicity of cells, as described in Zhao et al., J.Immunol., 174:4415-4423 (2005).

[0344] A biopsy is the removal of tissue and / or cells from an individual. Such removal may be for the purpose of collecting tissue and / or cells from the individual in order to perform experiments on the removed tissue and / or cells. The experiments may include experiments to determine whether the individual has and / or suffers from a particular condition or disease state. The condition or disease may be, for example, cancer. With regard to detecting the presence of cells expressing an anti-ADAM12 agent in a host, the sample containing the host's cells may be a sample containing whole cells, their lysates, or fractions of whole cell lysates, such as nuclear or cytoplasmic fractions, total protein fractions, or nucleic acid fractions. When the sample contains whole cells, the cells may be any cells of the host, for example, cells of any organ or tissue, including blood cells or endothelial cells.

[0345] Pharmaceutical Compositions The compositions of the present invention can be administered in a number of ways, depending on whether local or systemic treatment is desired.

[0346] In general, administration may be topical, parenteral or enteral.

[0347] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breakthrough of a subject's tissue and administration of the pharmaceutical composition through breakthrough in the tissue, thus generally resulting in direct administration into the bloodstream, into a muscle, or into an internal organ. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-permeable non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrathoracic, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial injection or infusion, as well as kidney dialysis infusion techniques. In a preferred embodiment, parenteral administration of the composition of the present invention includes subcutaneous or intraperitoneal administration.

[0348] The terms "oral," "intestinal," "enterally," "orally," "non-parenterally," "non-parenterally," and the like refer to the administration of a compound or composition to an individual by a route or manner along the digestive tract. Examples of "oral" routes of administration of a composition include, but are not limited to, swallowing the composition in liquid or solid form by mouth, administering the composition via a nasojejunal or gastrostomy tube, administering the composition intraduodenal, and administering rectally, e.g., using a suppository for the lower intestinal tract of the digestive tract.

[0349] The compositions of the present invention may be suitable for topical, parenteral, or enteral administration.

[0350] Preferably, the formulated composition comprising the Ab, antigen-binding Ab fragment, ADC, or CAR, a polynucleotide or vector encoding same, or a cell expressing same, is suitable for administration parenterally, e.g., via subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0351] Formulations of pharmaceutical compositions suitable for parenteral administration typically include the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further include one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9), although for some applications, the formulations may be more suitably formulated as sterile nonaqueous solutions or as dry forms in combination with a suitable vehicle, such as sterile, pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose. Such dosage forms may be suitably buffered, if desired. Other parenterally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.Such formulations can be made from biodegradable biocompatible polymers such as, for example, but not limited to, ethylene vinyl acetate, poly(alkyl cyanoacrylates), poly(anhydrides), poly(amindes), poly(esters), poly(ester amindes), poly(phosphoesters), polyglycolic acid (PGA), collagen, polyorthoesters, polylactic acid (PLA), poly(lactic-co-glycolidc acid) (PLAGA), or naturally occurring biodegradable polymers such as chitosan and hyaluronic acid based polymers (Kamaly N. et al, Chem Rev. Author manuscript, available in PMC 2017 Jul 13).

[0352] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, semisolids, single-phase compositions, multi-phase compositions (e.g., oil-in-water, water-in-oil), foams, microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like may be necessary or desirable.

[0353] Compositions and formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, Carder compounds, and other pharma- ceutically acceptable carriers or excipients.

[0354] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0355] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.

[0356] The pharmaceutical composition of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques known in the pharmaceutical industry.Such techniques include the step of combining active ingredient with pharmaceutical carrier or excipient.Generally, preparation is prepared by combining active ingredient with liquid carrier or finely divided solid carrier or both uniformly and intimately, and then, if necessary, shaping product.

[0357] The compositions of the present invention may be formulated to provide suitable in vivo distribution of the active ingredient. It is often difficult to concentrate the distribution of antitumor drugs at tumor sites, even when the drug has specificity for a molecule expressed by cancer cells. Various strategies have been developed to address this issue, and any suitable strategy may be applied to the present invention (e.g., as reviewed in Rosenblum D. et al., Nat Commun. 2018 Apr 12; 9(1): 1410. doi: 10.1038 / s41467-018-03705-y). To deliver a drug to the brain, the drug must cross the blood-brain barrier (BBB). Any suitable strategy that allows crossing the BBB may be utilized for the delivery of any of the anti-ADAM12 agents of the drug (e.g., for exemplary strategies, see Dong X. et al., Theranostics. 2018; 8(6): 1481-1493).

[0358] The compositions of the present invention may be formulated into any of many possible dosage forms, including, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, aerosols, and enemas.The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous, or mixed media.Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran.Suspensions may also contain stabilizers.

[0359] In one embodiment of the present invention, pharmaceutical compositions can be formulated and used as foams. Pharmaceutical foams include, but are not limited to, formulations such as emulsions, microemulsions, creams, jellies, and liposomes. These formulations have essentially similar properties, but differ in the consistency of ingredients and final products. Agents that enhance the uptake of oligonucleotides at the cellular level can also be added to the pharmaceutical compositions and other compositions of the present invention. For example, cationic lipids, such as lipofectin (US Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO97 / 30731), also enhance the cellular uptake of oligonucleotides.

[0360] The composition of the present invention may additionally contain other auxiliary components that are conventionally found in pharmaceutical compositions.Thus, for example, the composition may contain additional compatible pharmacoactive materials, such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials that are useful for the physical formulation of various dosage forms of the composition of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers.However, such materials, when added, should not unduly interfere with the biological activity of the components of the composition of the present invention.The formulation can be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring agents, flavorings and / or aromatic substances, etc.

[0361] The formulations comprising any of the anti-ADAM12 agents of the present invention or any of the cell aggregates expressing any of the anti-ADAM12 agents, such as the anti-ADAM CAR of the present invention, may comprise pharma- ceutically acceptable excipients. The excipients included in the formulation have different purposes, for example, depending on the CAR construct, the cell sub-aggregate used, and the mode of administration. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for infection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents, and lubricants. The formulations comprising the CAR-expressing cell aggregates of the present invention will typically be prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).

[0362] The formulation or composition may contain multiple active ingredients useful for the particular indication, disease, or condition treated with the binding molecule or cell, preferably those with complementary activities to the binding molecule or cell, and each activity does not adversely affect each other.Such active ingredients are preferably present in combination in amounts effective for the intended purpose.Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical active agents or drugs. Such an agent or drug may be, but is not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an antibacterial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an anti-fibrotic drug, an immunosuppressant drug, a steroid, a bronchodilator, a beta-blocker, a matrix metalloproteinase inhibitor, an ADAM12 inhibitor, an ADAM12 signaling inhibitor, an anti-ADAM12 agent of the present invention, an enzyme, a hormone, a neurotransmitter, a toxin, a chemical compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, a shRNA, a microRNA, an oligonucleotide, or an imaging drug. Specific examples include, but are not limited to, chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0363] In some embodiments, pharmaceutical compositions can use time-release, delayed release and sustained release delivery systems, so that the delivery of the composition is performed before the sensitization of the treated site and in sufficient time.Many types of release delivery systems are available and known.Such systems can avoid repeated administration of the composition, thereby increasing the convenience for subjects and physicians.

[0364] kit Also provided herein is a kit comprising (a) one or more of an anti-ADAM12 agent (Ab, antigen-binding Ab fragment, ADC, CAR), a polynucleotide encoding the same, a vector encoding the same, or a cell expressing the same, and (b) instructions for use, for example, in the treatment or diagnosis of a disease or condition associated with ADAM12. The kit may include a label indicating the intended use of the contents of the kit. As used herein, the term "label" includes any written, marketing, or recorded material provided with, with, in, or accompanying the kit.

[0365] Method of administration The route of administration used in the methods of the present invention may be any suitable route, depending on whether local or systemic treatment is desired.

[0366] In general, administration may be topical, parenteral or enteral.

[0367] Preferably, the formulated composition comprising an Ab, antigen-binding Ab fragment, ADC, or CAR, or a polynucleotide or vector encoding same, or a cell expressing same, may be administered parenterally, e.g., via subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0368] In the case of adoptive cell therapy, methods of administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions.For example, adoptive T cell therapy methods are described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al., U.S. Patent No. 4,690,915 to Rosenberg, Rosenberg (2011) Nat Rev Clin Oncol.8(10):577-85).See, for example, Themeli et al.(2013) Nat Biotechnol.31(10):928-933, Tsukahara et al.(2013) Biochem Biophys Res Commun 438(1):84-9, Davila et al.(2013) PLoS ONE 8(4):e61338.

[0369] In some embodiments, the compositions of the present invention may be administered using any suitable medical device (e.g., as reviewed in Richter BB, J. BioDrugs (2018) 32:425).

[0370] Dosage Regarding the administration of any of the anti-ADAM12 agents and compositions of the present invention, the dosage varies and depends, for example, on the target disease, the severity of the disease, the route of administration, and pharmacokinetic factors. The dosage can be modified based on the response observed in the subject.

[0371] For administration of either an anti-ADAM12 Ab, antigen-binding Ab fragment, or ADC, or a composition comprising same, an appropriate dosing regimen can be determined using any suitable methodology (e.g., Bai S. et al., Clin Pharmacokinet. 2012 Feb 1;51(2):119-35. doi:10.2165 / 11596370-000000000-00000).

[0372] In some embodiments, dosages can be from about 1 ng / kg to about 1 g / kg (of the subject's body weight) per day. In some aspects, doses can be from about 10 ng / kg / day to about 900 mg / kg / day, from about 20 ng / kg / day to about 800 mg / kg / day, from about 30 ng / kg / day to about 800 mg / kg / day, from about 40 ng / kg / day to about 700 mg / kg / day, from about 50 ng / kg / day to about 600 mg / kg / day, from about 60 ng / kg / day to about 500 mg / kg / day, from about 70 ng / kg / day to about 400 mg / kg / day, from about 80 ng / kg / day to about 300 mg / kg / day, from about 90 ng / kg / day to about 200 mg / kg / day, or from about 100 ng / kg / day to about 100 mg / kg / day. Treatment may be administered repeatedly or periodically to a subject for days, months, or years, or until a desired effect is achieved. An exemplary dosing regimen includes an initial dose of about 2 mg / kg of anti-ADAM12 Ab, antigen-binding Ab fragment, or ADC, followed by a maintenance dose of about 1 mg / kg administered once weekly.

[0373] Dosing frequency can be, for example, three times a day, twice a day, once a day, once every other day, once a week, once every other week, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every three months, once every four months, once every six months, once a year, or less frequently.

[0374] In some embodiments, the pharmaceutical composition contains cells expressing the CAR of the present invention in an amount effective for treating or preventing a disease or condition, for example, a therapeutically or prophylactically effective amount. In some embodiments, the therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the subject being treated. For repeated administration over several days or more, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosing regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple boluses of the composition, or by continuous infusion administration of the composition.

[0375] In certain embodiments, in the context of engineered cells expressing an anti-ADAM12 agent, such as a CAR, a subject may be administered, for example, a range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 50 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 1 trillion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or a range defined by any two of the foregoing values). cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the aforementioned values), in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges, and / or such number of cells per kilogram of the subject's body weight. For example, in some embodiments, administration of the cells or cell aggregates can include administration of about 103 to about 109 cells per kg of body weight, including all integer values ​​of the number of cells within these ranges.

[0376] The cells or cell aggregates may be administered in one or more doses. In some embodiments, the above-mentioned effective amount of cells may be administered as a single dose. In some embodiments, the above-mentioned effective amount of cells may be administered as two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the patient. The cells or cell aggregates may be obtained from any source, such as a blood bank or a donor. While individual needs vary, the determination of the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the age, health, and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect. In some embodiments, an effective amount of the cells or a composition comprising those cells is administered parenterally. In some embodiments, administration may be intravenous. In some embodiments, administration may be directly by injection at the site of the disease.

[0377] For purposes of the present invention, the amount or dose of the anti-ADAM12 material of the present invention administered should be sufficient to effect a therapeutic or prophylactic response in a subject or animal over a reasonable time frame. For example, the dose of the anti-ADAM12 material of the present invention should be sufficient to bind to an antigen or detect, treat, or prevent disease for a period of about 2 hours or more, e.g., about 12 to about 24 hours or more, from the time of administration. In certain embodiments, this period may be even longer. The dose will be determined by the efficacy of the particular anti-ADAM12 material of the present invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0378] For purposes of the present invention, the starting dose to be administered to a mammal can be determined using an assay that involves, for example, comparing the extent to which target cells are lysed, or in the context of a CAR, the extent to which IFN-γ is secreted by T cells expressing a CAR, polypeptide, or protein of the present invention upon administration of a given dose of T cells to the mammal, among a series of mammals each receiving a different dose of T cells. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a particular dose can be assayed by methods known in the art.

[0379] In some embodiments, two or more of the anti-ADAM12 agents or compositions of the invention may be administered to a subject, in combination or separately.

[0380] In some embodiments, the anti-ADAM12 agent or composition of the present invention is administered as part of a combination therapy, such as a cytotoxic agent or therapeutic agent, simultaneously with another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, or sequentially in any order. The cell or antibody in some embodiments is co-administered simultaneously with one or more additional therapeutic agents or in combination with another therapeutic intervention, simultaneously with one or more additional therapeutic agents, or sequentially in any order. In some contexts, the anti-ADAM12 agent or composition is administered simultaneously with another therapy close enough in time that the anti-ADAM12 agent or composition enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cell or antibody is administered before one or more additional therapeutic agents. In some embodiments, the anti-ADAM12 agent, such as an anti-ADAM12 CAR T cell or antibody, is administered after one or more additional therapeutic agents. In addition, the composition of the present invention can be given to a subject with one or more other therapies, which can be surgery or radiation therapy.

[0381] In some embodiments, in CAR T therapy, lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., injection) of CAR cells. In one example, lymphodepleting chemotherapy is administered to the subject prior to administration of the cells. For example, lymphodepleting chemotherapy is terminated 1-4 days (e.g., 1, 2, 3, or 4 days) prior to CAR cell injection. In some embodiments, multiple doses of CAR cells are administered, for example, as described herein. In some embodiments, lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., injection) of CAR-expressing cells described herein. Examples of lymphodepletion include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, and the like. Examples of lymphodepleting agents include, but are not limited to, anti-thymocyte globulin, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD52 antibodies, anti-CD2 antibodies, TCR alpha beta blockers, anti-CD20 antibodies, anti-CD19 antibodies, bortezomib, rituximab, anti-CD154 antibodies, rapamycin, CD3 immunotoxins, fludarabine, cyclophosphamide, busulfan, melphalan, MabThera, tacrolimus, alefacept, alemtuzumab, OKT3, OKT4, OKT8, OKT11, fingolimod, anti-CD40 antibodies, anti-BR3 antibodies, Campath-1H, anti-CD25 antibodies, calcineurin inhibitors, mycophenolate, and steroids, which may be used alone or in combination.

[0382] Use as a diagnostic tool The anti-ADAM12 agents of the invention, such as anti-ADAM12 Abs and antigen-binding Ab fragments, may also be useful as diagnostic tools, which may be used in vivo, ex vivo, or in vitro.

[0383] For example, anti-ADAM12 Ab or antigen-binding Ab fragment conjugated with imaging agent can be administered to subject or patient to test whether the patient's diseased cells or tissues express ADAM12.Diagnosis can be performed using any imaging tool that can detect imaging agent.Alternatively, a biological sample, such as, but not limited to, blood sample or biopsy sample, can be obtained, and anti-ADAM12 Ab or antigen-binding Ab fragment can be applied to the sample to test the expression of ADAM12.

[0384] These tests can determine whether the subject, or the subject's cells or tissues, express ADAM12. In some embodiments, the tests can determine whether the subject, or the subject's cells or tissues, express ADAM12 in sufficient amounts to be targeted by the anti-ADAM12 therapeutic agent of the present invention. In some embodiments, the tests can classify patients into different levels of ADAM12 expression. In one aspect, the subject can be classified as an expresser or a non-expresser. In another aspect, the subject can be classified as an overexpresser, an intermediate expresser, or a low expresser.

[0385] In some embodiments, the appropriate therapeutic approach may be determined depending on ADAM12 expression. Expression may be determined using the anti-ADAM12 agent of the present invention described herein, or alternatively, using any suitable method, such as, but not limited to, by measuring RNA expression levels, or by quantifying ADAM12 protein levels using suitable tools and / or techniques. In one aspect, the anti-ADAM12 agent of the present invention may be given to an expressor but not to a non-expressor. In another aspect, the anti-ADAM12 agent of the present invention may be given to an overexpressor but not to an intermediate or low expressor. In another aspect, the anti-ADAM12 agent of the present invention may be given to an overexpressor or intermediate expressor but not to a low expressor. In yet another aspect, the anti-ADAM12 agent of the present invention may be given to an intermediate expressor but not to a high or low expressor.

[0386] Variations The scope of the present invention includes functional portions of the anti-ADAM12 agents of the present invention described herein. The term "functional portion" when used in reference to an Ab, an antigen-binding Ab fragment, an ADC, or a CAR, refers to any portion or fragment of an Ab, an antigen-binding Ab fragment, an ADC, or a CAR of the present invention, which portion or fragment retains the biological activity of the Ab, an antigen-binding Ab fragment, an ADC, or a CAR of which it is a part (parent). Functional portions include, for example, those portions of an Ab, an antigen-binding Ab fragment, an ADC, or a CAR that retain the ability to recognize target cells or detect, treat, or prevent disease to a similar extent, to the same extent, or to a greater extent than the parent. With respect to a parent Ab, an antigen-binding Ab fragment, an ADC, or a CAR, a functional portion may, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent.

[0387] A functional portion can include additional amino acids at the amino or carboxy terminus of the portion, or at both termini, that are not found in the amino acid sequence of the parent Ab, antigen-binding Ab fragment, ADC, or CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, such as, for example, recognizing target cells, detecting, treating, or preventing fibrosis and / or inflammation. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent Ab, antigen-binding Ab fragment, ADC, or CAR.

[0388] The scope of the present invention includes functional variants of the Ab, antigen-binding Ab fragment, ADC, or CAR of the present invention described herein. The term "functional variant" as used herein refers to an Ab, antigen-binding Ab fragment, ADC, or CAR polypeptide or protein that has substantial or significant sequence identity or similarity with the parent of which the functional variant retains the biological activity of the Ab, antigen-binding Ab fragment, ADC, or CAR of which it is a variant. Functional variants include, for example, variants of the Ab, antigen-binding Ab fragment, ADC, or CAR (parent) described herein that retain the ability to recognize target cells to a similar, identical, or higher degree than the parent. With respect to a parent Ab, antigen-binding Ab fragment, ADC, or CAR, a functional variant can be, for example, at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical to the amino acid sequence of the parent.

[0389] A functional variant may, for example, comprise a parent amino acid sequence with at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant may comprise a parent amino acid sequence with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not prevent or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent.

[0390] The amino acid substitutions in the anti-ADAM12 agents of the invention are preferably conservative amino acid substitutions, which are known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain substituted with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituted with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid having a polar side chain substituted with another uncharged amino acid having a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid having a beta-branched side chain substituted with another amino acid having a beta-branched side chain (e.g., Ile, Thr, and Val), an amino acid having an aromatic side chain substituted with another amino acid having an aromatic side chain (e.g., His, Phe, Trp, Tyr, etc.), etc.

[0391] Also, amino acids can be added or removed from the sequence based on the vector design.

[0392] Anti-ADAM12 agents can consist essentially of a particular amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not substantially alter the biological activity of the functional variant.

[0393] The Abs, antigen-binding Ab fragments, ADCs, or CARs (including functional portions and functional variants) of the present embodiments can be of any length, i.e., can contain any number of amino acids, provided that the Abs, antigen-binding Ab fragments, ADCs, or CARs (or functional portions and functional variants thereof) retain their biological activity, e.g., the ability to specifically bind to an antigen, the ability to detect disease cells in a mammal, or the ability to treat or prevent disease in a mammal, etc. For example, the Abs, antigen-binding Ab fragments, ADCs, or CARs can be about 50 to about 5000 amino acids long, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids long.

[0394] The Ab, antigen-binding Ab fragment, ADC, or CAR of the present embodiments (including the functional portions and functional variants of the present invention) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline, and the like. N',N'-bibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropanoic acid, homophenylalanine, and α-tert-butylglycine.

[0395] The Abs, antigen-binding Ab fragments, ADCs, or CARs (including functional portions and functional variants) of the present embodiments may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted to acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0396] The Ab, antigen-binding Ab fragment, ADC, or CAR (including functional portions and functional variants thereof) of the present embodiments can be obtained by methods known in the art. The Ab, antigen-binding Ab fragment, ADC, and CAR can be produced by any suitable method for producing a polypeptide or protein. Suitable methods for de novo synthesis of polypeptides and proteins are described in references such as Chan et al., "Fmoc Solid Phase Peptide Synthesis", Oxford University Press, Oxford, United Kingdom, 2000, "Peptide and Protein Drug Analysis", ed. Reid, R., Marcel Dekker, Inc., 2000, "Epitope Mapping", ed. Westwood et al., "Oxford University Press, Oxford, United Kingdom, 2001, and U.S. Patent No. 5,449,752. Polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein using standard recombinant methods. See, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001, and Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing Associates and John Wiley & Sons, Inc., 2002. See, Sons, NY, 1994. Furthermore, some of the Abs, antigen-binding Ab fragments, or CARs (including functional parts and functional variants thereof) of the present invention can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, e.g., rats, humans, etc. Isolation and purification methods are known in the art. Alternatively, the Abs, antigen-binding Ab fragments, ADCs, or CARs (including functional parts and functional variants thereof) described herein can be commercially synthesized by companies.In this regard, the Abs, antigen-binding Ab fragments, ADCs, or CARs of the invention can be synthesized, recombinant, isolated, and / or purified.

[0397] definition Although various embodiments and examples of the invention have been described with reference to particular molecules, compositions, methods, or protocols, it should be understood that the invention is not limited to the particular molecules, compositions, methods, or protocols described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular aspects or embodiments only, and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0398] All references cited herein, including patent and non-patent literature, are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0399] In the above specification and the appended claims, all transitional phrases such as "comprising," "including," "having," "containing," "involving," "consisting of," and the like, are understood to be open-ended, i.e., meant to include, but not be limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0400] It should also be noted that, unless the context dictates otherwise, the singular forms "a," "an," and "the" as used herein and in the appended claims include plural references. Thus, reference to a "cell" refers to one or more cells and equivalents thereof known to those of skill in the art, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0401] Unless expressly indicated otherwise, in any method disclosed or claimed herein that includes two or more steps, the order of steps performed should be understood to be not limited by the order of the steps recited.

[0402] The terms "4-1BB", "41BB", or "BB" as used herein refer to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA53133.1, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one aspect, the "4-1BB costimulatory domain", also referred to as the "4-1BB CS domain" or "41BBCS", may be derived from the cytoplasmic domain of 4-1BB. In some embodiments, the "41BBCS" comprises the sequence provided as SEQ ID NO: 165, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In some embodiments, the "41BBCS" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 265.

[0403] The term "5' cap" as used herein (also referred to as RNA cap, RNA 7-methylguanosine cap or RNA m7G cap) is a modified guanine nucleotide added to the "front" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group linked to the first transcribed nucleotide. Its presence is important for recognition by ribosomes and protection from RNases. Capping is coupled to transcription and occurs co-transcriptionally, so that each affects the other. Immediately after transcription initiation, the 5' end of the synthesized mRNA is bound by a cap synthesis complex that associates with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate the functionality of the mRNA, such as its stability or translation efficiency.

[0404] The term "about" or "approximately" as used herein when referring to numerical values ​​such as weight, mass, volume, concentration, or time, should not be limited to the recited numerical values ​​but encompasses a variation of + / - 10% of the given value.

[0405] As used herein, "ADAM12" is also known as ADAM metallopeptidase domain 12, a disintegrin and metalloproteinase-12, a disintegrin and metalloproteinase domain-containing protein 12, ADAM12-OT1, CAR10, MCMP, MCMPMltna, Meltrin-a, MLTN, or MLTNA, and is a member of the ADAM family (a disintegrin and metalloproteinase family). In humans, ADAM12 is encoded by the ADAM12 gene on chromosome 10, with gene location 10q26.2 (NCBI), and there are two naturally occurring ADAM12 splice variants named ADAM12-L (L for long) and ADAM12-S (S for short) (Kveiborg M. et al., Int J Biochem Cell Biol. 2008; 40 (9): 1685-702. doi: 10.1016 / j.biocel.2008.01.025. Epub 2008 Feb 1). Human ADAM12-L has the amino acid sequence provided as GenBank accession: AAC08702.2, or the equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, human ADAM12-L has the amino acid sequence provided as SEQ ID NO: 101, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). Human ADAM12-S has the amino acid sequence provided as GenBank Accession: AAC08703.2, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, human ADAM12-S has the amino acid sequence provided as SEQ ID NO: 102, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

[0406] The term "allogeneic" as used herein refers to any material that is derived from a different animal of the same species as the individual to which the material is introduced.Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical.In some embodiments, allogeneic materials from individuals of the same species may not be genetically similar enough to interact antigenically.

[0407] The term "antibody" or "Ab" or "immunoglobulin" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. Typically, a full-size Ab contains two pairs of chains, each pair containing a heavy chain (HC) and a light chain (LC). The HC typically contains a variable region and a constant region. The LC also typically contains a variable region and a constant region. The variable region of the heavy chain (VH) typically contains three complementarity determining regions (CDRs), referred to herein as CDR 1, CDR 2, and CDR 3 (or referred to as CDR-H1, CDR-H2, and CDR-H3, respectively). The constant region of the HC typically contains the fragment crystallizable region (Fc region), which determines the isotype of the Ab, the type of Fc receptor to which the Ab binds, and thus the effector function of the Ab. Types of Fc receptors include, but are not limited to, FcaR (e.g., FcaRI), Fca / mR, FceR (e.g., FceRI, FceRII), and FcgR (e.g., FcgRI, FcgRIIA, FcgRIIB1, FcgRIIB2, FcgRIIIA, FcgRIIIB), and their associated downstream effectors are well known in the art. The variable region of the light chain (VL) also typically comprises CDRs, which are CDR 1, CDR 2, and CDR 3 (or referred to as CDR-L1, CDR-L2, CDR-L3, respectively). In some embodiments, the antigen is ADAM12. The antibody may be an intact immunoglobulin derived from a natural or recombinant source. The portion of the antibody that contains the structure that allows it to specifically bind to the antigen is referred to as the "antigen-binding fragment," "AB domain," "antigen-binding region," or "AB region" of the Ab.

[0408] The term "antibody-drug conjugate," "Ab-drug conjugate," or "ADC" as used herein refers to a conjugate of an Ab or an antigen-binding Ab fragment with a drug. The drug may be connected to any portion of the Ab or antigen-binding Ab fragment via direct or indirect conjugation, such as via a linker. In some embodiments, the ADC may include an antibody (or an antibody fragment, such as a single chain variable fragment (scFv)) linked to a payload drug (often cytotoxic). The antibody binds the ADC to the target cancer cell. In some embodiments, the ADC is then internalized by the cell and the drug is released intracellularly. Because of the targeting, side effects may be even lower and may provide a wider therapeutic window. A hydrophilic linker (e.g., PEG4Mal) may prevent the drug from being pumped out of resistant cancer cells via MDR (multidrug resistance) transporters. The present disclosure also relates to immunoconjugates comprising anti-ADAM12 binding agents conjugated to therapeutic agents such as cytotoxins, drugs (e.g., immunosuppressants) or radiotoxins. Such conjugates may be referred to as "immunoconjugates". Immunoconjugates comprising one or more cytotoxins may be referred to as "immunotoxins". Cytotoxins or cytotoxic agents include any agent that is detrimental to (e.g., kills) cells. Cytotoxins may be conjugated to the antibodies of at least some embodiments of the present invention using linker technology available in the art. Examples of linker types that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Linkers may be selected that are susceptible to cleavage, for example, by the low pH in the lysosomal compartment or by proteases, such as proteases preferentially expressed in tumor tissues, such as cathepsins (e.g., cathepsins B, C, D).For further discussion of the types of linkers and methods for conjugating cytotoxins, therapeutic agents to antibodies, see Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, PA et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, TM (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, RJ (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter, PD and Springer, CJ (2001) Adv. Drug Deliv. Rev. 53:247-264. The antibodies of the present invention can also be conjugated to radioisotopes to generate cytotoxic radiopharmaceuticals (also called radioimmunoconjugates).

[0409] The term "antibody fragment" or "Ab fragment" as used herein refers to any portion or fragment of an Ab, including intact or full-length Abs that may be of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The term encompasses molecules constructed using one or more portions or fragments of one or more Abs. An Ab fragment may be an immunoreactive portion of an intact immunoglobulin. The term is used in its broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments (including single chain variable fragments (scFv)), diabodies, and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFvs, tandem tri-scFvs. In certain embodiments, the antibody fragment is an scFv.

[0410] Unless otherwise indicated, the term "Ab fragment" should be understood to encompass functional antibody fragments thereof. The portion of an Ab fragment that contains the structure that enables specific binding to an antigen is referred to as the "antigen-binding Ab fragment," "AB domain," "antigen-binding region," or "antigen-binding region" of the Ab fragment.

[0411] As used herein, Ab "heavy chain" or "HC" refers to the larger of the two polypeptide chains present in all Ab molecules in their naturally occurring conformations.

[0412] As used herein, the "light chain" or "LC" of an Ab refers to the smaller of the two polypeptide chains present in all Ab molecules in their naturally occurring conformations. Kappa light chains and lambda light chains refer to the two major antibody light chain isotypes.

[0413] "Anti-ADAM12 agent" or "anti-ADAM12 material" as used herein refers to any agent that can directly or indirectly target ADAM12.Anti-ADAM12 agents of the present invention include, but are not limited to, anti-ADAM12 Ab, anti-ADAM12 antigen-binding Ab fragment, anti-ADAM12 multispecific Ab, anti-ADAM12 multispecific antigen-binding Ab fragment, anti-ADAM12 ADC, anti-ADAM12 CAR, and the nucleic acid sequences and vectors encoding them, and the cells expressing them.In a broad sense, anti-ADAM12 agents can also include pharmaceutical compositions that contain any of the above-mentioned anti-ADAM12 agents.

[0414] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, cancer tissue samples, tumor tissue samples, leukemia cell samples, inflamed tissue samples, as well as cells or fluids with other biological components. In some embodiments, the antigen is ADAM-12.

[0415] The term "antigen binding domain" or "AB domain" refers to a portion of an anti-ADAM12 agent of the present invention, which portion comprises a structure that allows the anti-ADAM12 agent to specifically bind to ADAM12. When the anti-ADAM12 agent is an Ab, the AB domain can comprise a variable region or a portion of the variable region (e.g., CDR) of the Ab. When the anti-ADAM12 agent is an antigen-binding Ab fragment or an antibody-drug conjugate, the AB domain can comprise a variable region or a portion of the variable region (e.g., CDR) of the Ab from which the anti-ADAM12 agent is derived. When the anti-ADAM12 agent is a chimeric antigen receptor (CAR), the AB domain can be one or more extracellular domains of the CAR with specificity for ADAM12. When the AB domain is derived from an Ab or an antigen-binding Ab fragment, the AB domain can comprise a variable region or a portion of the variable region, e.g., CDR, of the Ab or antigen-binding Ab fragment from which the AB domain is derived. In some embodiments, the AB domain of the anti-ADAM12 agent of the present invention is an scFv. In some embodiments, the AB domain may comprise or be derived from a naturally occurring molecule that binds to ADAM12 or the ADAM12-binding portion of the molecule. Examples of such molecules include, but are not limited to, alpha actinin 2 (ACTN2), insulin-like growth factor binding protein 3 (IGFBP3), phosphatidylinositol 3 kinase regulatory subunit alpha (PIK3R1), IGFBP-3 (insulin-like growth factor binding protein 3), IGFBP-5, heparin-binding epidermal growth factor (HB-EGF), epidermal growth factor (EGF), betacellulin, delta-like 1, placental leucine aminopeptidase (P-LAP), and matrix metalloprotease 14 (MMP-14).

[0416] The term "apheresis," as used herein, refers to the art-recognized extracorporeal process in which a donor's or patient's blood is removed from the donor's or patient's blood and passed through an apparatus that separates selected specific components and returns the remainder to the donor's or patient's circulation, e.g., by retransfusion. Thus, in the context of an "apheresis sample," refers to a sample obtained using apheresis.

[0417] As used herein, the terms "autologous" or "donor-derived" refer to any material derived from the same individual into which it is later reintroduced.

[0418] The term "binding" refers to an attractive interaction between two molecules that results in a stable association when the molecules are in close proximity to one another. The result of molecular binding is sometimes the formation of a molecular complex in which the attractive forces holding the components together are generally non-covalent and therefore usually energetically weaker than covalent bonds.

[0419] The term "cancer" refers to a disease characterized by uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers related to the present invention include, but are not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, small cell lung cancer, gastric cancer, or thyroid cancer.

[0420] As used herein, the term "bispecific" refers to having two binding specificities. For example, the anti-ADAM12 bispecific Ab or bispecific antigen-binding Ab fragment of the present invention has at least one specificity for ADAM12. If the first specificity is specific to an epitope for ADAM12, the second specificity may be specific to another non-overlapping or non-competing epitope of ADAM12, or may be specific to a molecule other than ADAM12. The term "bispecific" is used in the same way for any other anti-ADAM12 agent of the present invention, such as anti-ADAM12 CAR.

[0421] The term "CD28" refers to the protein cluster of differentiation 28, one of the proteins expressed on T cells that provides costimulatory signals required for T cell activation and survival. Human CD28 protein may have at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI reference number NP_006130 or a fragment thereof with stimulatory activity. The term "CD28 transmembrane domain," also referred to as "CD28 TM domain" or "CD28TM," refers to amino acid residues derived from the transmembrane domain of CD28. In some embodiments, "CD28TM" comprises the sequence provided as SEQ ID NO: 161, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In some embodiments, the "CD28 TM domain" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 261. The term "CD28 hinge," as used herein, refers to amino acid residues that may be used in some embodiments of the CAR to link two domains or two portions within the domain. In some embodiments, the "CD28 hinge" comprises the sequence provided as SEQ ID NO: 163, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In some embodiments, the "CD28 hinge" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 263. The term "CD28 costimulatory domain", also referred to as "CD28 CS domain" or "CD28CS", refers to amino acid residues derived from the cytoplasmic domain of CD28. In some embodiments, the "CD28CS" comprises the sequence provided as SEQ ID NO: 164, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In some embodiments, the "CD28 CS domain" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 264.

[0422] The term "CD3 zeta", or alternatively "zeta", "zeta chain", "CD3-zeta", "CD3z", "TCR-zeta", or "CD247", is a protein that in humans is encoded by the CD247 gene on chromosome 1 with gene location 1q24.2. CD3 zeta forms the TCR complex together with the T cell receptor (TCR) and CD3 (a protein complex composed of CD3 gamma, CD3 delta, and two CD3 epsilons). Human CD3 zeta may have the amino acid sequence provided as NP_000725 or NP_932170, or equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.). The term "CD3 zeta intracellular signaling domain", or alternatively "CD3 zeta ICS domain" or "CD3zICS", is defined as amino acid residues from the cytoplasmic domain of the CD3 zeta chain sufficient to functionally transmit the initial signal required for T cell activation, or a functional derivative thereof. In one embodiment, the "CD3 zeta ICS domain" is the sequence provided as SEQ ID NO: 162. In one embodiment, the "CD3 zeta ICS domain" is encoded by the nucleic acid sequence provided as SEQ ID NO: 262.

[0423] The term "chimeric antigen receptor", or alternatively "CAR", refers to a set of polypeptides, typically two in the simplest embodiment, that in the case of an immune effector cell, confer specificity to the cell for a target cell and direct intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain (AB domain), a transmembrane domain (TM domain), and an intracellular signaling domain (also referred to herein as an "intracellular signaling domain (ICS domain)") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous to one another. In some embodiments, the set of polypeptides comprises a dimerization switch that allows the polypeptides to bind to one another, e.g., the AB domain to bind to the ICS domain, in the presence of a dimerization molecule. In one aspect, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic portion of the CAR further comprises a costimulatory domain (CS domain) that comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), DAP10 and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular AB domain, a TM domain, and an ICS domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular AB domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and a CS domain comprising a functional signaling domain derived from a costimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular AB domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and two CS domains each comprising two functional signaling domains derived from costimulatory molecules that are the same as or different from each other.In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular AB domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and at least two CS domains each comprising a functional signaling domain derived from a costimulatory molecule that is the same as or different from each other. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, which is optionally cleaved from the antigen binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. In some embodiments, the leader sequence (LS) comprises the amino acid sequence provided as SEQ ID NO: 160. In some embodiments, the LS can be encoded by a nucleic acid sequence provided as SEQ ID NO: 260.

[0424] The term "competes" as used herein with respect to an Ab, antigen-binding Ab fragment of an AB domain of any of the anti-ADAM12 agents of the invention means that a first Ab, antigen-binding Ab fragment, or AB domain binds to an epitope in a manner sufficiently similar to the binding of a second Ab, antigen-binding Ab fragment, or AB domain, such that the result of binding of the first Ab, antigen-binding Ab fragment, or AB domain to its cognate epitope is detectably reduced in the presence of the second Ab, antigen-binding Ab fragment, or AB domain compared to binding of the first Ab, antigen-binding Ab fragment, or AB domain in the absence of the second Ab, antigen-binding Ab fragment, or AB domain. Alternatively, binding of the second Ab, antigen-binding Ab fragment, or AB domain to its epitope could also be detectably reduced in the presence of the first antibody, but this is not necessarily the case. That is, a first Ab, antigen-binding Ab fragment, or AB domain may inhibit the binding of a second Ab, antigen-binding Ab fragment, or AB domain to its epitope without the second Ab, antigen-binding Ab fragment, or AB domain inhibiting the binding of the first Ab, antigen-binding Ab fragment, or AB domain to its respective epitope. However, if each Ab, antigen-binding Ab fragment, or AB domain detectably inhibits the binding of the other Ab, antigen-binding Ab fragment, or AB domain to its cognate epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, the two (Abs, antigen-binding Ab fragments, or AB domains) are said to "cross-compete" with each other for their respective epitopes. Both competing and cross-competing Abs, antigen-binding Ab fragments, or AB domains are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope, or portions thereof), one of skill in the art will understand, based on the teachings provided herein, that such competing and / or cross-competing Abs, antigen-binding Ab fragments, or AB domains are encompassed and useful in the methods disclosed herein.

[0425] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and refer to non-contiguous sequences of amino acids within an antibody variable region, and are known in the art to confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3).

[0426] The term "conservative amino acid substitution" as used herein is as commonly used in the art and includes amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain substituted with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituted with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid having a polar side chain substituted with another uncharged amino acid having a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid having a beta-branched side chain substituted with another amino acid having a beta-branched side chain (e.g., Ile, Thr, and Val), an amino acid having an aromatic side chain substituted with another amino acid having an aromatic side chain (e.g., His, Phe, Trp, Tyr, etc.), etc. A non-conservative amino acid substitution is an amino acid substitution that is not a conservative amino acid substitution.

[0427] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, Toll ligand receptors, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5, CD7, CD8 alpha, CD8 beta, CD11a, LFA-1 ( CD11a / CD18), CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84 , CD96(Tactile), CD100(SEMA4D), CD103, OX40(CD134), 4-1BB(CD137), SLAM(SLAMF1, CD150, IPO-3), CD160(BY55), S ELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITG B1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, ​​NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, and a ligand that specifically binds CD83. In embodiments where the CAR comprises one or more CS domains, each CS domain comprises a functional signaling domain derived from a costimulatory molecule.In some embodiments, the encoded CS domain comprises 4-1BB, CD28, or DAP10. In one embodiment, the CS domain comprises the amino acid sequence of CD28CS, 41BBCS, or DAP10CS (SEQ ID NO: 164, 165, or 166), or is encoded by the encoded nucleotide sequence provided as SEQ ID NO: 264, 265, or 266.

[0428] The term "cytokine" as used herein refers to a broad category of small proteins involved in cell signaling. Generally, their release has some effect on the behavior of cells in their surroundings. Cytokines may participate in autocrine signaling, paracrine signaling and / or endocrine signaling as immunomodulatory agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, epithelial cells, and various stromal cells. "Chemokines" are a family of cytokines generally involved in mediating chemotaxis.

[0429] The term "cytotoxicity" generally refers to any cell killing activity caused by exposure of the anti-ADAM12 agent of the present invention or cells containing it to cells expressing ADAM12. This activity can be measured by known cytotoxicity assays, including IFN-γ production assays. When the target cell is a cancer or tumor cell, the term "anti-cancer cytotoxicity" or "anti-tumor cytotoxicity" can be used.

[0430] The term "DAP10" refers to a protein encoded by the HSCT gene in humans. It may also be referred to as HSCT, KAP10, PIK3AP, or hematopoietic cell signal transducer. In some embodiments, DAP10 may have the sequence provided in Genbank Accession No. Q9UBK5.1. The term "DAP10 costimulatory domain", also referred to as "DAP10 CS domain" or "DAP10CS", refers to amino acid residues derived from the cytoplasmic domain of DAP10. In some embodiments, "DAP10CS" comprises the sequence provided as SEQ ID NO: 166, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In some embodiments, the "DAP10 CS domain" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 266.

[0431] The phrase "diseases associated with expression of ADAM12" or "ADAM12-associated diseases" includes, but is not limited to, diseases associated with expression of ADAM12, including proliferative diseases such as cancer or malignant tumors or precancerous conditions, or conditions associated with cells expressing ADAM12, or non-cancer-related indications associated with cells expressing ADAM12. Non-cancer-related indications associated with ADAM12 include fibrosis, autoimmunity, cardiovascular conditions, allergic conditions, respiratory diseases, nephropathy, neurological diseases, muscular diseases, liver diseases, metabolic syndrome, infections, and inflammatory disorders. Examples of various cancers that express ADAM12 include, but are not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumors, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, and thyroid cancer.

[0432] "Effective amount" or "therapeutically effective amount" refers to a dose sufficient to prevent or treat a disease, condition, or disorder in an individual. The amount effective for therapeutic or prophylactic use will depend, for example, on the stage and severity of the disease or disorder being treated, the age, weight, and general health of the patient, other pre-existing conditions, and the judgment of the prescribing physician. The size of the dose will also be determined by the selected active ingredient, the method of administration, the timing and frequency of administration, the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular active ingredient, and the desired physiological effect. Those skilled in the art will understand that various diseases or disorders may require long-term treatment with multiple administrations, possibly using the anti-ADAM12 agents, nucleic acids, vectors, cells, or compositions of the present invention, at each or various rounds of administration.

[0433] The terms "enteral," "enterally," "oral," "orally," "non-parenterally," "non-parenterally," and the like refer to administration of a compound or composition to an individual by a route or manner along the digestive tract. Examples of "oral" routes of administration of a composition include, but are not limited to, swallowing the composition in liquid or solid form by mouth, administering the composition via a nasojejunal or gastrostomy tube, administering the composition intraduodenal, and administering rectally, e.g., using a suppository for the lower intestinal tract of the digestive tract.

[0434] The term "framework" as used herein refers to the non-CDR portion of the variable region of an Ab, or in some embodiments, the antigen-binding Ab fragment or AB domain of a CAR. "Heavy chain (HC) framework" refers to the non-CDR portion of the HC variable region, and generally there are four framework regions (FRs) (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region. "Light chain (LC) framework" refers to the non-CDR portion of the LC variable region, and generally there are four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. In some embodiments, the "human-like HC framework" is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human HC framework. In some embodiments, a "human-like LC framework" is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human LC framework.

[0435] The term "gene" is used broadly to refer to any segment of polynucleotide related to biological function. Thus, a gene includes introns and exons as in genomic sequences, or simply coding sequences as in cDNA, and / or regulatory sequences required for their expression. For example, a gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or codes for a specific protein, and includes regulatory sequences.

[0436] The terms "hinge," "spacer," or "linker" refer to an amino acid sequence of variable length that is typically encoded between two or more domains or portions of a polypeptide construct to confer flexibility, improved spatial organization, proximity, and the like.

[0437] As used herein, "human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or that is produced using any of the techniques for producing human antibodies known in the art or disclosed herein. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, human antibodies are selected from a phage library, which expresses human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be produced by immunization of animals in which human immunoglobulin loci have been transgenic in place of endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies directed against a target antigen (such B lymphocytes can be recovered from an individual or from single cell cloning of cDNA, or immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147 (1):86-95, 1991; and U.S. Patent No. 5,750,373.

[0438] The term "humanization" of an Ab refers to modifying an Ab of non-human origin to increase sequence similarity with Abs naturally produced in humans. As used herein, the term "humanized antibody" refers to an Ab generated through humanization of an Ab. Generally, a humanized or engineered antibody has one or more amino acid residues from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically obtained from an "import" variable, constant, or other domain of a known human sequence. Known human Ig sequences are disclosed, for example, at www.ncbi.nlm.nih.gov / entrez / query.fcgi; www.atcc.org / phage / hdb.html, each of which is incorporated herein by reference in its entirety. Such import sequences can be used to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. Generally, some or all of the non-human or human CDR sequences are maintained, while some or all of the non-human sequences of the framework and / or constant regions are replaced with human or other amino acids. Antibodies can also be optionally humanized while retaining high affinity for antigens and other favorable biological properties, using three-dimensional immunoglobulin models known to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, framework (FR) residues can be selected and combined from the consensus and import sequences to achieve desired antibody properties, such as increased affinity for the target antigen. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.Humanization or engineering of the antibodies of the present invention can be carried out using methods such as, but not limited to, those described by Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151:2296 (1993), Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992), Presta et al. al., J. Immunol. 151:2623 (1993), U.S. Patent No. 5,723,323, U.S. Patent No. 5,976,862, U.S. Patent No. 5,824514, U.S. Patent No. 5,817 No. 483, No. 5,814476, No. 5,763,192, No. 5,723,323, No. 5,766,886, No. 5,714,352, No. 6,204,0 This can be done using any known method, such as those described in US Pat. Nos. 23, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, and 4,816,567 (each of which is incorporated by reference in its entirety, including the references cited therein).

[0439] The term "iCAR" refers to a chimeric antigen receptor that contains an inhibitory receptor signaling domain. These domains can be based on, for example, Protectin D1 (PD1) or CTLA-4 (CD152). In some embodiments, the CAR-expressing cells of the present invention are further transduced to express an iCAR. In one aspect, the iCAR is added to restrict the functional activity of the CAR-expressing cells to tumor cells.

[0440] The term "immune cell" refers to a cell of hematopoietic origin that is functionally involved in the initiation and / or execution of innate and / or adaptive immune responses.

[0441] The term "intracellular signaling domain" or "ICS domain" as used herein refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of cells transduced with a nucleic acid sequence, including a CAR, e.g., a CAR T cell. For example, examples of immune effector function in CAR T cells include cytolytic activity and helper activity, including secretion of cytokines. ICS domains include the ICS domain of lymphocyte receptor chain, TCR / CD3 complex protein, Fc receptor subunit, IL-2 receptor subunit, CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, CD278 (ICOS), Fc epsilon RI, DAP10, and DAP12.

[0442] An "isolated" biological component (e.g., an isolated protein, nucleic acid, vector, or cell) refers to a component that has been substantially separated or purified from its environment or other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles) within the cell of the organism in which it naturally occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins that have been purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant techniques and chemical synthesis. An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell.

[0443] A "leader sequence" or "LS" as used herein, also referred to in the art as a "signal peptide", "signal sequence", "targeting signal", "localization signal", "localization sequence", "transit peptide" or "leader peptide", is a short peptide present at the N-terminus of most newly synthesized proteins that are directed to the secretory pathway. The core of the signal peptide may contain a long stretch of hydrophobic amino acids. The signal peptide may or may not be cleaved from the mature polypeptide.

[0444] The term "linker" as used in the context of ScFv refers to a peptide linker consisting of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy and variable light regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises one or more repeats of the amino acid sequence unit Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 167). In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)3, also referred to as G4S X3 (SEQ ID NO: 168). Such a linker may be encoded, for example, by the nucleic acid sequence set forth in SEQ ID NO: 268.

[0445] The term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, e.g., mice, rats, and hamsters, and mammals of the order Logomorpha, e.g., rabbits. The mammal may be a mammal from the order Carnivora, including Feline (cat) and Canine (dog). The mammal may be a mammal from the order Artiodactyla, including Bovine (cow) and Swine (pig), or Perssodactyla, including Equine (horse). The mammal may be a mammal from the orders Primate, Ceboid, or Simoid (monkeys), or Anthropoid (humans and apes).

[0446] The term "masked CAR" refers to a CAR-expressing cell that further comprises a masking peptide. This masking peptide can prevent off-target cell killing. The masking peptide is often N-terminal to the CAR construct and can block the cell's ability to bind to unintended targets. The masking peptide can be cleaved from the CAR-expressing cell when it encounters a tumor, allowing the CAR-expressing cell to attack its target without killing off-target cells. The anti-ADAM12 CAR of the present invention can be constructed to be a masked CAR.

[0447] As used herein, the term "multispecific" refers to having two or more binding specificities.For example, the anti-ADAM12 multispecific Ab or multispecific antigen-binding Ab fragment of the present invention has at least one specificity for ADAM12.If the first specificity is specific to an epitope for ADAM12, the second (or third, fourth, etc.) specificity may be specific to another epitope for ADAM12, or may be specific to a molecule other than ADAM12.The term "multispecific" is used in the same way for any other anti-ADAM12 agent of the present invention, such as anti-ADAM12 CAR.

[0448] The terms "nucleic acid" and "polynucleotide" refer to RNA or DNA that is linear or branched, single-stranded or double-stranded, or a hybrid thereof. The terms also encompass RNA / DNA hybrids. The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, for example, by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more analogs of naturally occurring nucleotides, and the introduction of a means for attaching the polynucleotide to a protein, a metal ion, a labeling component, another polynucleotide, or a solid support. The polynucleotides may be obtained by chemical synthesis or may be derived from a microorganism.

[0449] The term "OKT3" or "muromonab-CD3" or "orthoclone OKT3" refers to a monoclonal anti-CD3 Ab.

[0450] The term "parenteral" or "parenteral" as used herein includes any route of administration of a compound or composition characterized by physical breakthrough of a subject's tissue, and administration of a pharmaceutical composition through breakthrough in tissue, thus generally resulting in direct administration into the bloodstream, into muscle, or into an internal organ. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-permeable non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrathoracic, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial injection or infusion, as well as kidney dialysis infusion techniques. In a preferred embodiment, parenteral administration of the composition of the present invention includes subcutaneous or intraperitoneal administration.

[0451] The terms "pharmaceutical acceptable excipient", "pharmaceutical excipient", "excipient", "pharmaceutical acceptable carrier", "pharmaceutical carrier", or "carrier" as used herein refer to compounds or materials typically used in pharmaceutical compositions to facilitate formulation and / or storage.

[0452] The term "promoter," as used herein, is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate specific transcription of a polynucleotide sequence.

[0453] "Ri...

Claims

1. an antibody (Ab) or antigen-binding Ab fragment, wherein the Ab or antigen-binding Ab fragment binds to ADAM12; (a) a heavy chain (HC) variable domain, HC complementarity determining region 1 (CDR-H1), HC complementarity determining region 2 (CDR-H2), HC complementarity determining region 3 (CDR-H3), and a HC variable domain comprising a human-like HC framework; (b) a light chain (LC) variable domain, LC complementarity determining region 1 (CDR-L1), LC complementarity determining region 2 (CDR-L2), LC complementarity determining region 3 (CDR-L3), and a LC variable domain comprising a human-like LC framework; Optionally, the Ab or Ab fragment is selected from the group consisting of a monoclonal Ab, a monospecific Ab, a bispecific Ab, a multispecific Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain specific Ab, a domain deleted Ab, a scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, a CDR grafted Ab, a fragment antigen binding (Fab), an F(ab')2, an Fab' fragment, a variable fragment (Fv), a single chain Fv (scFv) fragment, an Fd fragment, a diabody, and a minibody.

2. (i) the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively, and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences set forth in SEQ ID NOs: 136, 137, and 138, respectively; or (ii) the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences encoded by SEQ ID NOs:232, 233, and 234, respectively, and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences encoded by SEQ ID NOs:236, 237, and 238, respectively; or (iii) the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences set forth in SEQ ID NOs: 142, 143, and 144, respectively, and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences set forth in SEQ ID NOs: 146, 147, and 148, respectively; or (iv) the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences encoded by SEQ ID NOs:242, 243, and 244, respectively, and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences encoded by SEQ ID NOs:246, 247, and 248, respectively; Optionally, (a) the human-like HC framework is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human HC framework; (b) the human-like LC framework is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human LC framework.

3. (i) the HC variable domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 131 and the LC variable domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135; or (ii) the HC variable domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:231, and the LC variable domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:235; or (iii) the HC variable domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 141, and the LC variable domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 145; or (iv) the Ab or antigen-binding Ab fragment of any one of claims 1-3, wherein the HC variable domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:241, and the LC variable domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:

245.

4. (i) an amino acid sequence set forth in SEQ ID NO: 139, 140, 149 or 150; or (ii) The Ab or antigen-binding Ab fragment of any one of claims 1-3, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:239, 240, 249, or 250.

5. two or more binding specificities, a first specificity being specific for an epitope of ADAM12; (i) the second specificity is for another epitope of ADAM12, or (ii) the second specificity is for an epitope of a second antigen other than ADAM12, optionally selected from the group consisting of CD3, NKG2D, 4-1BB, and Fc receptor (FcR).

6. comprising a human-like fragment crystallizable (Fc) region; (i) optionally, the human-like Fc region is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region; and (ii) optionally further comprising: the human-like Fc Region binds to an FcR selected from the group consisting of Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIB1, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha / mu receptor (Fca / mR), and neonatal Fc receptor (FcRn).

7. An antibody-drug conjugate (ADC), comprising: (a) the Ab or antigen-binding Ab fragment of any one of claims 1 to 6; (b) a drug conjugated to the Ab or antigen-binding Ab fragment; (i) optionally, the drug is an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an antibacterial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an antifibrotic drug, an immunosuppressant drug, a steroid, a bronchodilator, a beta-blocker, a matrix metalloproteinase inhibitor, an ADAM12 inhibitor, an ADAM12 signaling inhibitor, an enzyme, a hormone, a neurotransmitter, a toxin, a radioisotope, a chemical compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, shRNA, microRNA, an oligonucleotide, or an imaging drug; (ii) further optionally, the drug is doxorubicin, daunorubicin, cucurbitacin, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, nemorubicin, cryptophycin, menthacarcin, ansamitocin, mitomycin C, geldanamycin, mekkerucalmycin, rebeccamycin, safracin, okilactomycin, oligomycin, actinomycin, sandramycin, hypothemycin, polyketomycin, tomycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, taltoburin, lactacystin, dolastatin, auristatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), telomestatin, tubastatin A, combretastatin, maytansinoids, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodiazepines, SN-38, Ro 5-3335, pwainaphycin, duocarmycin, bafilomycin, taxoid, tubulysin, ferulenol, luciol A, fumagillin, hygrolysin, glucopyericidin, amanitin, ansatrienin, sinerubin, phallacidin, phalloidin, phytosphongosine, piericidin, poronetin, fodophyllotoxin, gramicidin A, sanguinarine, sinefungin, herboxidiene, microcorrin B, microcystin, muscotoxin A, tritoxin, an ADC selected from the group consisting of sine, triporin A, myoseverin, mitoxin B, nocuolin A, pseudodoralic acid B, pseudolotin A, cyclopamine, curbulin, colchicine, aphidicolin, englerin, cordycepin, apoptolidin, epothilone A, limaquinone, isatropolone, isofistularin, quinaldopeptin, ixabepilone, aeroprisinin, arginosin, agrochelin, epothilone, and derivatives of any one of the above.

8. A chimeric antigen receptor (CAR), (a) an antigen-binding (AB) domain that binds to ADAM12; (b) a transmembrane (TM) domain; and (c) an intracellular signaling (ICS) domain; and (d) optionally a hinge connecting the AB domain and the TM domain; (e) optionally, one or more costimulatory (CS) domains.

9. The AB domain is an Ab or antigen-binding Ab fragment according to any one of claims 1 to 6, Optionally, (I) the AB domain, (i) an amino acid sequence set forth in SEQ ID NO: 139, 140, 149 or 150; or (ii) comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:239, 240, 249, or 250; (II) the AB domain, (i) an amino acid sequence set forth in SEQ ID NO: 139, 140, 149 or 150; or (ii) competes for binding to ADAM12 with an scFv comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 239, 240, 249, or 250; or (III) the AB domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an ADAM12 binding domain of a naturally occurring ADAM12 binding molecule; Further optionally, the native ADAM12-binding molecule is selected from the group consisting of alpha actinin 2 (ACTN2), insulin-like growth factor binding protein 3 (IGFBP3), IGFBP5, phosphatidylinositol 3 kinase regulatory subunit alpha (PIK3R1), heparin-binding epidermal growth factor (HB-EGF), epidermal growth factor (EGF), betacellulin, delta-like 1, placental leucine aminopeptidase (P-LAP), and matrix metalloprotease (MMP-14).

10. The TM domain is (I) derived from the TM region or a transmembrane portion thereof of a protein selected from the group consisting of CD28, CD3e, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCRa, TCRb, and CD3z; (II) optionally derived from the TM region of CD28 or a transmembrane portion thereof; (III) further optionally, (i) the amino acid sequence set forth in SEQ ID NO: 161, or (ii) the CAR of claim 8 or 9, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:

261.

11. The ICS domain is (I) derived from a cytoplasmic signaling sequence of a protein selected from the group consisting of CD3z, lymphocyte receptor chain, TCR / CD3 complex protein, Fc receptor (FcR) subunit, IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, and DAP12, or a functional fragment thereof; (II) optionally derived from the cytoplasmic signaling sequence of CD3z, or a functional fragment thereof; (III) further optionally, (i) the amino acid sequence set forth in SEQ ID NO: 162, or (ii) The CAR of any one of claims 8 to 10, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:

262.

12. The hinge is (I) derived from CD28; (II) optionally, (i) the amino acid sequence set forth in SEQ ID NO: 163, or (ii) The CAR according to any one of claims 8 to 11, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:

263.

13. At least one of the one or more CS domains: (I) CD28, DAP10, 4-1BB (CD137), CD2, CD4, CD5, CD7, CD8a, CD8b, CD11a, CD11b, CD11c, CD11d, CD18 , CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103 , OX40 (CD134), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 ( CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CEACAM1, CDS, CR TAM, GADS, GITR, HVEM (LIGHTER), IA4, ICAM-1, IL2Rb, IL2Rg, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, ​​NKG2C, NKG2D, NKp30, derived from a cytoplasmic signaling sequence of a protein selected from the group consisting of NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, and CD83 ligand, or a functional fragment thereof; (II) optionally derived from a cytoplasmic signaling sequence of CD28, 4-1BB, or DAP10, or a functional fragment thereof; (III) further optionally, (i) the amino acid sequence set forth in SEQ ID NO: 164; (ii) the amino acid sequence encoded by SEQ ID NO: 264; (iii) the amino acid sequence set forth in SEQ ID NO: 165; (iv) the amino acid sequence encoded by SEQ ID NO: 265; (v) the amino acid sequence set forth in SEQ ID NO: 166, or (vi) The CAR according to any one of claims 8 to 12, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:

266.

14. (i) the amino acid sequence of h6E6scFvHL-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 171); (ii) the amino acid sequence of h6E6scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 172); (iii) the amino acid sequence of h6E6scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 173); (iv) the amino acid sequence of h6E6scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 174); (v) the amino acid sequence of h6E6scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 175); (vi) the amino acid sequence of h6E6scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 176); (vii) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 177); (viii) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 178); (ix) the amino acid sequence of h6C10scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 179); (x) the amino acid sequence of h6C10scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 180); (xi) the amino acid sequence of h6C10scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 181); (xii) the amino acid sequence of h6C10scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 182); or (xiii) The CAR of any one of claims 8 to 13, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by any one of SEQ ID NOs: 271 to 282.

15. The CAR of any one of claims 8 to 14, further comprising a cytotoxic drug conjugated to the AB domain.

16. An isolated nucleic acid sequence encoding the antibody (Ab) or antigen-binding Ab fragment of any one of claims 1 to 6.

17. An isolated nucleic acid sequence encoding the CAR of any one of claims 8 to 15.

18. moreover (I) a leader sequence (LS), optionally at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:260, and optionally (II) The isolated nucleic acid sequence of claim 17, comprising a T2A sequence and / or a sequence encoding a truncated CD19 (trCD19), wherein the T2A sequence is optionally at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 269, and the trCD19 optionally comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:

170.

19. A vector comprising a nucleic acid sequence according to any one of claims 16 to 18, Optionally, said vector is selected from a DNA, RNA, plasmid, cosmid, viral vector, lentiviral vector, adenoviral vector, or retroviral vector.

20. 1. A recombinant or isolated cell comprising: (i) the Ab or antigen-binding Ab fragment of any one of claims 1 to 6; (ii) the ADC of claim 7; (iii) The CAR according to any one of claims 8 to 15. (iv) a nucleic acid sequence according to any one of claims 16 to 18, or (v) comprising the vector of claim 19, Optionally, the cells are (I) a non-mammalian cell, optionally selected from the group consisting of a plant cell, a bacterial cell, a fungal cell, a yeast cell, a protozoan cell, and an insect cell; (II) a mammalian cell, optionally selected from the group consisting of a human cell, a rat cell, and a mouse cell; (III) Stem cells, (IV) primary cells, optionally human primary cells, or cells derived therefrom; (V) a cell line, optionally a hybridoma cell line; (VI) immune cells, (VII) MHC+ or MHC-; or (VIII) cell lines, T cells, T cell precursors, CD4+ T cells, helper T cells, regulatory T cells, CD8+ T cells, naive T cells, effector T cells, memory T cells, stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, cytotoxic T cells, mucosal associated invariant T (MAIT) cells, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, and a / b T cells, g / d A recombinant or isolated cell selected from the group consisting of T cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, perforin-deficient cells, granzyme-deficient cells, B cells, myeloid cells, monocytes, macrophages, and dendritic cells.

21. The cell expresses the CAR of any one of claims 8 to 15, and optionally the cell has an endogenous T cell receptor (TCR) that is (i) is not expressed, (ii) is not functionally expressed, or (iii) a T cell that is engineered to express a reduced level relative to a wild-type T cell; Further optionally, when the CAR binds to its target molecule: (I) the cells are activated or stimulated to proliferate; (II) the cell exhibits cytotoxicity against a cell expressing the target molecule; (III) administering the cells ameliorates the disease, cancer, cardiac condition, autoimmune condition, inflammatory condition, or fibrotic condition; (IV) the cells increase expression of a cytokine and / or a chemokine, optionally wherein the cytokine is IFN-g; or (V) the cell has reduced expression of a cytokine and / or chemokine, optionally wherein the cytokine is TGF-b.

22. 22. A collection of cells comprising at least one first recombinant or isolated cell of claim 20 or 21 and, optionally, at least one second recombinant or isolated cell engineered to express a different CAR, ADC, or antibody or antigen-binding antibody fragment than the first recombinant or isolated cell.

23. 1. A pharmaceutical composition comprising: (a)(i) the Ab or antigen-binding Ab fragment of any one of claims 1 to 6; (ii) the ADC of claim 7; (iii) The CAR according to any one of claims 8 to 15. (iv) a nucleic acid sequence according to any one of claims 16 to 18, (v) the vector according to claim 19 , (vi) a cell according to claim 20 or 21, or (vii) a cell aggregate according to claim 22; (b) optionally, a pharma- ceutically acceptable excipient or carrier.

24. 1. A method of treating a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of (i) the Ab or antigen-binding Ab fragment of any one of claims 1 to 6; (ii) the ADC of claim 7; (iii) The CAR according to any one of claims 8 to 15. (iv) a nucleic acid sequence according to any one of claims 16 to 18, (v) the vector according to claim 19 , (vi) a cell according to claim 20 or 21, (vii) a population of cells according to claim 22; or (viii) administering the pharmaceutical composition of claim 23, Optionally, the method further comprises: (I) cancer, fibrosis, autoimmunity, cardiovascular conditions, allergic conditions, respiratory diseases, nephropathy, neurological diseases, muscular diseases, liver diseases, metabolic syndrome, infectious or inflammatory disorders, or (II) The method for use in treating cancer, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, and thyroid cancer.

25. 1. A method of treating a subject with an anti-ADAM12 agent, the method comprising: (a) obtaining or obtaining a biological sample from said subject; (b) measuring the expression level of ADAM12 in the biological sample; (c) determining whether the subject is an ADAM12 overexpressor; (d) administering to the subject a therapeutically effective amount of (i) the Ab or antigen-binding Ab fragment of any one of claims 1 to 6; (ii) the ADC of claim 7; (iii) The CAR according to any one of claims 8 to 15. (iv) a nucleic acid sequence according to any one of claims 16 to 18, (v) the vector according to claim 19 , (vi) a cell according to claim 20 or 21, (vii) a population of cells according to claim 22; or (viii) administering the pharmaceutical composition of claim 23, Optionally, the subject is suffering from cancer, and the cancer is optionally selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, and thyroid cancer.

26. 1. A method for stimulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of (i) the Ab or antigen-binding Ab fragment of any one of claims 1 to 6; (ii) the ADC of claim 7; (iii) The CAR according to any one of claims 8 to 15. (iv) a nucleic acid sequence according to any one of claims 16 to 18, (v) the vector according to claim 19 , (vi) a cell according to any one of claims 20 or 21, (vii) a population of cells according to claim 22; or (viii) A method comprising administering the pharmaceutical composition of claim 23.

27. A method of treating a disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (i) the Ab or antigen-binding Ab fragment of any one of claims 1 to 6; (ii) the ADC of claim 7; (iii) The CAR according to any one of claims 8 to 15. (iv) a nucleic acid sequence according to any one of claims 16 to 18, (v) the vector according to claim 19 , (vi) a cell according to claim 20 or 21, (vii) a population of cells according to claim 22; or (viii) administering the pharmaceutical composition of claim 23, The disease is (I) cancer, fibrosis, autoimmunity, cardiovascular conditions, allergic conditions, respiratory diseases, nephropathy, neurological diseases, muscular diseases, liver diseases, metabolic syndrome, infectious or inflammatory disorders, or (II) a cancer selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, and thyroid cancer; (III) or any combination of the above.

28. 1. A method of expanding a population of cells in a subject, the method comprising: (i) a nucleic acid sequence according to any one of claims 16 to 18, (ii) the vector according to claim 19 , (iii) a cell according to claim 20 or 21, (iv) a population of cells according to claim 22; or (v) administering the pharmaceutical composition of claim 23, (I) optionally, the resulting population of cells is maintained in the subject for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 2 years, or at least 3 years following administration; (II) Optionally, the subject is suffering from cancer, and the cancer is further optionally selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, esophagogastric adenocarcinoma, oligodendroglioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, gastric cancer, and thyroid cancer.

29. 29. The method of any one of claims 24-28, further comprising administering a second agent, wherein the second agent is optionally an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an antibacterial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an antifibrotic drug, an immunosuppressant drug, a steroid, a bronchodilator, a beta-blocker, a matrix metalloproteinase inhibitor, an ADAM12 inhibitor, an ADAM12 signaling inhibitor, an enzyme, a hormone, a neurotransmitter, a toxin, a radioisotope, a chemical compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, shRNA, microRNA, an oligonucleotide, an imaging drug, or a CAR, ADC, or an antibody or antigen-binding antibody fragment that targets an antigen other than ADAM12, or a recombinant or isolated cell engineered to express a CAR, ADC, or an antibody or antigen-binding antibody fragment that targets an antigen other than ADAM12.

30. A method for generating a cell comprising the CAR according to any one of claims 8 to 15, the method comprising: (i) introducing into a cell (ia) a nucleic acid sequence encoding at least one CAR according to any one of claims 8 to 15, or (ib) at least one nucleic acid sequence according to claim 17 or 18; or (ii) transducing a cell with the vector of claim 19; Optionally, (iii) isolating the cells based on expression of the CAR and / or a selectable marker as determined via flow cytometry or immunofluorescence assay.

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