CN109562172B - Efficacy of anti-HLA-DR antibody drug conjugate IMMU-140(hL243-CL2A-SN-38) in HLA-DR positive cancers - Google Patents

Abstract

The present invention relates to therapeutic immunoconjugates comprising SN-38 linked to an anti-HLA-DR antibody or antigen-binding antibody fragment. The immunoconjugate may be administered at a dose of between 3mg/kg and 18mg/kg, preferably 4,6, 8,9, 10, 12, 16 or 18mg/kg, more preferably 8, 10 or 12 mg/kg. When administered at the indicated doses and schedules, the immunoconjugates can reduce the size of solid tumors, reduce or eliminate metastases, and are effective in treating cancers that are resistant to standard treatments such as radiation therapy, chemotherapy, or immunotherapy. The methods and compositions are particularly useful for treating AML, ALL, or multiple myeloma.

Description

Efficacy of anti-HLA-DR antibody drug conjugate IMMU-140(hL243-CL2A-SN-38) in HLA-DR positive cancers

Cross Reference to Related Applications

This application is a continuation-in-part application of U.S. patent application serial No. 15/281,453 filed on 30/9/2016, U.S. patent application serial No. 15/281,453 is a division of U.S. patent application serial No. 14/667,982 (now U.S. patent No. 9,493,573) filed on 25/3/2015, U.S. patent application serial No. 14/667,982 is a division of U.S. patent application serial No. 13/948,732 (now U.S. patent No. 9,028,833) filed on 23/7/2013, U.S. patent application serial No. 13/948,732 claims the benefit of provisional U.S. patent application serial No. 61/749,548 filed on 7/2013/2012 and provisional U.S. patent application serial No. 61/736,684 filed on 13/12/2012. This application is a continuation-in-part application of U.S. patent application serial No. 15/484,308 filed on 11/4/2017, U.S. patent application serial No. 15/484,308 claiming the benefit of provisional U.S. patent application serial No. 62/373,591 filed on 11/2016 and provisional U.S. patent application serial No. 62/322,441 filed on 14/4/2016, according to 35u.s.c.119 (e). This application claims the benefit of provisional U.S. patent application serial No. 62/373,591 filed on day 11/8/2016 and provisional U.S. patent application serial No. 62/428,231 filed on day 30/11/2016 in 35u.s.c.119 (e). The entire contents of each priority application are incorporated herein by reference.

Sequence listing

This application contains a sequence listing, which has been filed in ASCII format via EFS-Web and is incorporated by reference herein in its entirety. The ASCII copy created on 25.7.7.2017 was named IMM369WO1_ sl. txt, size 9,661 bytes.

Technical Field

The present invention relates to therapeutic uses of immunoconjugates of an antibody or antigen-binding antibody fragment and a camptothecin (e.g., SN-38), said conjugates having improved ability to target a variety of cancer cells in a human subject. Preferably, the antibody or fragment is an anti-HLA-DR antibody or fragment, such as hL 243. In other preferred embodiments, the antibody and therapeutic moiety are linked by an intracellularly cleavable linkage, which increases therapeutic efficacy. In some more preferred embodiments, the immunoconjugate is administered at a specific dose and/or specific administration regimen that optimizes the therapeutic effect. The optimized dose and administration regimen of the SN-38 conjugated anti-HLA-DR antibody for human therapeutic use disclosed herein shows unexpectedly superior efficacy not predicted from animal model studies, allowing for effective treatment of cancers that are resistant to standard anti-cancer therapies, including the parent compound irinotecan (CPT-11). The immunoconjugate may be administered alone or in combination with one or more other therapeutic agents administered prior to, concurrently with, or subsequent to the immunoconjugate. Exemplary therapeutic agents that may be used in combination with anti-HLA-DR include, but are not limited to, proteasome inhibitors such as bortezomib, Bruton kinase inhibitors such as ibrutinib, or phosphoinositide-3-kinase inhibitors such as idelalisib. In some preferred embodiments, the cancer to be treated is an HLA-DR positive cancer, such as B-cell lymphoma, B-cell leukemia, skin cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, endometrial cancer, cervical cancer, testicular cancer, melanoma, renal cancer, or liver cancer. More preferably, the cancer is AML (acute myeloid leukemia), ALL (acute lymphocytic leukemia) or MM (multiple myeloma). Most preferably, the patient to be treated has relapsed from or shows resistance to at least one standard anti-cancer therapy prior to treatment with the immunoconjugate. However, one of ordinary skill will recognize that in some embodiments, the immunoconjugate may be used for first line therapy.

Background

For many years, scientists in the field of specifically targeted drug therapy have aimed the use of monoclonal antibodies (mabs) to specifically deliver toxic agents to human cancers. Conjugates of tumor-associated mabs and suitable toxic agents have been developed, but have had half-way success in human cancer therapy and have little application to other diseases, such as autoimmune diseases. The most common toxic agents are chemotherapeutic drugs, although particle emitting radionuclides or bacterial or phytotoxins are also conjugated to MAbs, particularly for the treatment of Cancer (Sharkey and Goldenberg, CA Cancer J Clin.2006 Jul-Aug; 56(4): 226-.

The advantage of using MAb-chemotherapeutic drug conjugates is that (a) the chemotherapeutic drug itself is well defined in structure; (b) chemotherapeutic drugs are attached to MAb proteins, usually at specific sites remote from the antigen binding region of the MAb, using well-defined conjugation chemistry; (c) MAb-chemotherapeutic drug conjugates can be made more reproducibly and are generally less immunogenic than chemical conjugates involving mabs and bacterial or phytotoxins, and are therefore easier to develop commercially and to regulatory approve; and (d) MAb-chemotherapeutic drug conjugates are orders of magnitude less in systemic toxicity than radionuclide MAb conjugates, particularly in radiation-sensitive bone marrow.

Camptothecin (CPT) and its derivatives are a class of potent antitumor agents. Irinotecan (also known as CPT-11) and topotecan are CPT analogs, which are approved Cancer therapeutics (Iyer and rainin, Cancer chemother. pharmacol.42: S31-S43 (1998)). CPT acts by stabilizing The topoisomerase I-DNA complex to inhibit topoisomerase I (Liu et al: underfolding Their anticerancer Potential, Liehr J.G., Giovanella, B.C. and Verschraegen (eds.), NY Acad Sci., NY 922:1-10 (2000)). CPT presents particular problems in the preparation of conjugates. One problem is the insolubility of most CPT derivatives in aqueous buffers. Secondly, CPT provides specific challenges for structural modification directed to conjugation to macromolecules. For example, CPT itself contains only tertiary hydroxyl groups in ring-E. In the case of CPT, the hydroxyl functionality must be coupled to a linker suitable for subsequent protein conjugation; and in potent CPT derivatives, such as SN-38, the active metabolite of the chemotherapeutic agent CPT-11, and other C-10-hydroxy-containing derivatives such as topotecan and 10-hydroxy-CPT, the presence of the phenolic hydroxy group at the C-10 position complicates the necessary derivatization of the C-20-hydroxy group. Third, the instability of the delta-lactone moiety of the E-ring of camptothecin under physiological conditions leads to a substantial reduction in antitumor efficacy. Thus, the conjugation scheme is performed such that it is performed at a pH of 7 or lower to avoid lactone ring opening. However, conjugation of bifunctional CPTs with amine reactive groups (e.g. active esters) typically requires a pH of 8 or higher. Fourth, the intracellularly cleavable moiety is preferably incorporated into a linker/spacer that links the CPT and the antibody or other binding moiety.

Human leukocyte antigen-DR (HLA-DR) is one of three isoforms of Major Histocompatibility Complex (MHC) class II antigens. HLA-DR is highly expressed in a variety of hematological malignancies and has been actively used in antibody-based Lymphoma therapy (Brown et al, 2001, Clin Lymphoma 2: 188-90; DeNardo et al, 2005, Clin Cancer Res 11:7075s-9 s; Stein et al, 2006, Bloood 108: 2736-44). Human HLA-DR antigens are expressed at levels significantly higher than typical B cell markers, including CD20, in non-hodgkin's lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL), and other B cell malignancies. Preliminary studies have shown that anti-HLA-DR mAbs are significantly more potent than other naked mAbs currently of clinical interest in vitro and in vivo experiments with lymphomas, leukemias, and multiple myeloma (Stein et al, unpublished results).

HLA-DR is also expressed on a subset of normal immune cells, including B cells, monocytes/macrophages, Langerhans cells, dendritic cells and activated T cells (Dechant et al, 2003, Semin Oncol 30: 465-75). Therefore, it may not be surprising that previous attempts to develop anti-HLA-DR antibodies were hampered by toxicity, particularly infusion-related toxicity that may be associated with complement activation (Lin et al, 2009, Leuk Lymphoma 50: 1958-63; Shi et al, 2002, Leuk Lymphoma 43: 1303-12).

The L243 antibody (hereinafter referred to as mL243) is a murine IgG2a anti-HLA-DR antibody. By targeting the D region of HLA, the antibodies can potentially be used to treat diseases such as autoimmune diseases or cancer, particularly leukemia or lymphoma. mL243 showed effective inhibition of immune function in vitro and was monomorphic for all HLA-DR proteins. However, there is a problem in administering mouse antibodies to human patients, such as inducing a human anti-mouse antibody (HAMA) response. There is a need for more effective compositions and methods of use of anti-HLA-DR antibodies with improved efficacy and reduced toxicity. There is also a need for more efficient methods of making and administering antibody-CPT conjugates, such as anti-HLA-DR-SN-38 conjugates. Preferably, the methods include optimized dosages and administration regimens that maximize the efficacy and minimize toxicity of the antibody-CPT conjugates for therapeutic use in human patients.

Disclosure of Invention

The abbreviation "CPT" as used herein may refer to camptothecin or any derivative thereof, e.g., SN-38, unless explicitly stated otherwise. The present invention addresses an unmet need in the art by providing improved methods and compositions for preparing and administering CPT-antibody immunoconjugates. Preferably, the camptothecin is SN-38. The disclosed methods and compositions may be used to treat a variety of diseases and disorders that are difficult to treat with or less responsive to other forms of therapy, and may include suitable antibodies or antigen-binding antibody fragments that selectively target them, which may be developed, available or known diseases, such as cancer.

Preferably, the targeting moiety is an antibody, antibody fragment, bispecific antibody or other multivalent antibody, or other antibody-based molecule or compound. More preferably, the antibody or fragment is an anti-HLA-DR antibody or fragment. The antibody may be of various isotypes, preferably human IgG1, IgG2, IgG3 or IgG4, more preferably comprising human IgG1 hinge and constant region sequences. Most preferably, the antibody is human IgG 4. The antibody or fragment thereof may be a chimeric human-mouse antibody, a chimeric human primate antibody, a humanized (human framework and murine hypervariable (CDR) regions) antibody or a fully human antibody, and variants thereof, such as a half IgG4 antibody (referred to as a "mono antibody"), as described by van der Neut Kolfschoeten et al (Science 2007; 317: 1554) 1557. More preferably, the antibody or fragment thereof may be designed or selected to comprise a human constant region sequence belonging to a particular allotype, which may result in reduced immunogenicity when the immunoconjugate is administered to a human subject. Preferred allotypes for administration include non-G1 m1 allotypes (nG1m1), such as G1m3, G1m3,1, G1m3,2, or G1m3,1, 2. More preferably, the allotype is selected from the group consisting of nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

Where the disease state is cancer, a number of antigens expressed by or otherwise associated with tumor cells are known in the ART, including, but not limited to, carbonic anhydrase IX, alpha-fetoprotein (AFP), alpha-actinin-4, A, antigens specific for A antibodies, ART-4, B, Ba 733, BAGE, BrE-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL, CD1, CD11, CD32, CD40, CD 66-e, CD70, CD79, CD126, CD132, CD133, CD147, CD154, CDK-4/m, CTD-4, CDK 2-CDC, KN-4-CDC, CD-4, CD-CDK 4, CDK, CD-CDK 4, CD-CDK-4, CD-CDK, CD-1, CD-1, CD-CD, CD-1, CD-CD, CD-1, CD-4, CD-CD, CD-4, CD-CD, CD-CD, CD-4, CD-CD, CD-4, CD-CD, CD-CD, CD-CD, CD-4, CD-CD, CD-4, CD-1, CD-C, CD-1, CD-C, CD-1, CD-4, CD-C, CD-1, CD-, CXCR4, CXCR7, CXCL12, HIF-1 alpha, colon specific antigen-p (CSAP), CEA (CEACAM5), CEACAM6, c-Met, DAM, DLL3, DLL4, EGFR, EGFRvIII, EGP-1(TROP-2), EGP-2, ELF2-M, Ep-CAM, Fibroblast Growth Factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-beta, HLA-DR, HM1.24, Human Chorionic Gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-gamma, IFN-alpha, IFN-beta, IFN-lambda, IL-4-R, IL-R, IL-13-R, IL-15-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC 4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage Migration Inhibitory Factor (MIF), MAGE-3, MART-1, MART-2, mesothelin, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, pancreatic cancer mucin, PD-1 receptor, growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF- α, Tn antigen, Thomson-Friedenich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factor C3, C3a, C3B, C5a, C5, angiogenic markers, bcl-2, bcl-6, Kras, oncogene markers, and oncogene products (see, e.g., Sensi et al, Clin Cancer Res 2006, Immunor 12: 5023-32; Parmiani et al, J2007,178: 1975-187; Canmunol et al, Cancer Nomuro et al, 2005: 69). Preferably, the antibody binds to CEACAM5, CEACAM6, EGP-1(TROP-2), MUC-16, AFP, MUC5ac, CD74, CD19, CD20, CD22 or HLA-DR. Preferred anti-HLA-DR antibodies can be used alone or in combination with another anti-TAA (tumor associated antigen) antibody.

Exemplary antibodies that can be used include, but are not limited to, hR1 (anti-IGF-1R, U.S. Pat. No. 9,441,043), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20 (anti-CD 20, U.S. Pat. No. 7,151,164), hA19 (anti-CD 19, U.S. Pat. No. 7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1 (anti-CD 74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD 22, U.S. Pat. No. 5,789,554), hMu-9 (anti-CSAP, U.S. Pat. No. 7,387,772), hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM 5, U.S. Pat. No. 6,676,924), hMN-15 (anti-CEACAM 6, U.S. Pat. No. 8,287,865), hRS7 (anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM 6, U.S. Pat. No. 7,541,440), Ab124, and Ab125 (anti-CXCR 4, U.S. Pat. No. 7,138,496), the examples of each referenced patent or application being incorporated herein in part by reference. More preferably, the antibody is IMMU-31 (anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti-CEACAM 5), hMN-3 (anti-CEACAM 6), hMN-15 (anti-CEACAM 6), hLL1 (anti-CD 74), hLL2 (anti-CD 22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD 19) or hA20 (anti-CD 20). Most preferably, the antibody is hL 243. As used herein, the terms epratuzumab (epratuzumab) and hLL2 are interchangeable, as are the terms veltuzumab (veltuzumab) and hA20, and the terms hL243g4P, hL243 γ 4P and IMMU-114.

Alternative antibodies used include, but are not limited to, abciximab (abciximab) (anti-glycoprotein IIb/IIIa), alemtuzumab (alemtuzumab) (anti-CD 52), bevacizumab (bevacizumab) (anti-VEGF), cetuximab (cetuximab) (anti-EGFR), gemtuzumab (gemtuzumab) (anti-CD 33), ibritumomab (ibritumomab) (anti-CD 20), panitumumab (anti-EGFR), rituximab (rituximab) (anti-CD 20), tositumomab (tositumomab) (anti-CD 20), trastuzumab (trastuzumab) (anti-ErbB 2), pembrolizumab (pemolizumab) (anti-PD-1 receptor), nivolumab (nivolumab) (anti-PD-1 receptor), ipilimumab (ipilimumab) (anti-4), CTLA (avamab (abagavacab) (anti-PD-125) (anti-PD-IL-125), adalimumab (anti-IL-125), rituximab (anti-IL-4), and the like (abagab) (anti-125), and the like (anti-IL-receptor (anti-IL-receptor) Obinutuzumab (obinutuzumab) (GA101, anti-CD 20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patent application 11/983,372, accession nos. ATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575), toslizumab (anti-IL-6 receptor), basiliximab (anti-CD 25), daclizumab (daclizumab) (anti-CD 25), efuzumab (anti-CD 11a), GA101 (anti-CD 20; Glycart Roche), molol-CD 3 (mucomonab-CD 3) (anti-CD 3 receptor), natalizumab (natalizumab) (anti- α 4 integrin), omalizumab (anti-CD 3 receptor); anti-TNF-. alpha.antibodies, such as CDP571(Ofei et al, 2011, Diabetes 45:881-85), MTNFAI, M2TNFAI, M3 TNFAAI, M3TNFABI, M302B, M303(Thermo Scientific, Rockford, IL), infliximab (infliximab) (Centocor, Malvern, PA), certolizumab (certolizumab pegol) (UCB, Brussels, Belgium), anti-CD 40L (UCB, Brussels, Belgium), adalimumab (adalimumab) (Abbott, Abbott Park, IL) or belimumab (Benlysta) (Human Genome Sciences).

In a preferred embodiment, the chemotherapeutic moiety is selected from Camptothecin (CPT) and analogues and derivatives thereof, more preferably SN-38. However, other chemotherapeutic moieties that may be used include taxanes (e.g., baccatin III (baccatin III), paclitaxel), epothilones (epothilones), anthracyclines (e.g. epirubicin (DOX), epirubicin, morpholino-DOX, cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolinoddoxorubicin (2-PDOX) or prodrug forms of 2-PDOX (pro-2-PDOX); see, e.g., Priebe W (eds.), ACS symposium series 574, published by American Chemical Society, Washington D.C., 1995(332pp) and Nagy et al, Proc. Natl. Acad. Sci. USA 93:2464-2469,1996), benzoquinone ansamycin (benzoquinoid ansamycin), e.g. geldanamycin (Bordean et al, Journal of Antibiotics 23: 447, 1970; Neckers et al, drug 17: 373, 1999). Preferably, the antibody or fragment thereof is linked to at least one chemotherapeutic moiety; preferably 1 to about 5 chemotherapeutic moieties; more preferably 6 or more chemotherapeutic moieties, more preferably 7 to 8, most preferably about 6 to about 12 chemotherapeutic moieties.

An example of a water-soluble CPT derivative is CPT-11. Extensive clinical data are available regarding the pharmacology of CPT-11 and its conversion to active SN-38 in vivo (Iyer and Ratain, Cancer Chemother Pharmacol.42: S31-43 (1998); Mathijssen et al, Clin Cancer Res.7: 2182-one 2194 (2002); Rivory, Ann NY Acad Sci.922: 205-one 215, 2000)). The active form SN-38 is about 2 to 3 orders of magnitude more potent than CPT-11. In certain preferred embodiments, the immunoconjugate may be an hMN-14-SN-38, hMN-3-SN-38, hMN-15-SN-38, IMMU-31-SN-38, hRS7-SN-38, hA20-SN-38, hL243-SN-38, hLLl-SN-38, or hLL2-SN-38 conjugate.

Various embodiments may relate to the treatment of cancer using the methods and compositions of the present invention, including but not limited to non-hodgkin's lymphoma, B cell acute and chronic lymphocytic leukemias, Burkitt's lymphoma, hodgkin's lymphoma, acute large B cell lymphoma, hairy cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, T cell lymphoma and leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, cancer, melanoma, sarcoma, glioma, bone and skin cancer. The cancer may include oral cancer, esophageal cancer, gastrointestinal cancer, respiratory cancer, lung cancer, gastric cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, bladder cancer, pancreatic cancer, bone cancer, brain cancer, connective tissue cancer, liver cancer, gallbladder cancer, bladder cancer, kidney cancer, skin cancer, central nervous system cancer, and testicular cancer.

In certain embodiments involving cancer treatment, the drug conjugates can be used in combination with surgery, radiation therapy, chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy, immunomodulators, vaccines and the like. Such combination therapy may allow lower doses of each therapeutic agent to be administered in such combinations, thereby reducing certain serious side effects, and possibly reducing the course of therapy required. When there is no or minimal overlapping toxicity, the entire dose of each may also be administered.

Preferred optimal doses of the immunoconjugate may comprise a dose of between 3mg/kg and 18mg/kg, more preferably between 4 and 16mg/kg, more preferably between 6 and 12mg/kg, most preferably between 8 and 10mg/kg, preferably administered weekly, twice weekly or every other week. An optimal dosing regimen may include the following treatment cycles: two consecutive treatment weeks followed by one, two, three or four rest weeks, or alternating treatment weeks and rest weeks, or one treatment week followed by two, three or four rest weeks, or three treatment weeks followed by one, two, three or four rest weeks, or four treatment weeks followed by one, two, three or four rest weeks, or five treatment weeks followed by one, two, three, four or five rest weeks, or once every two weeks, once every three weeks, or once every month. The treatment may extend over any number of cycles, preferably at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, or at least 16 cycles. Exemplary dosages for use may include 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg and 18 mg/kg. One of ordinary skill will recognize that in selecting the optimal dose of immunoconjugate, a variety of factors may be considered, such as age, general health, specific organ function or weight, and the effect of prior treatment on a specific organ system (e.g., bone marrow), and the dose and/or frequency of administration may be increased or decreased during treatment. When evidence of tumor shrinkage is observed after as little as 4 to 8 doses, the dose can be repeated as needed. The optimal dosages and administration regimens disclosed herein exhibit unexpectedly superior efficacy and reduced toxicity in human subjects, which is unpredictable in animal model studies. Surprisingly, the superior efficacy allows for the treatment of tumors previously found to be resistant to one or more standard anti-cancer treatments, including the parent compound CPT-11, from which SN-38 was derived in vivo.

A surprising result of utilizing the presently claimed compositions and methods is the unexpected tolerability of high dose antibody-drug conjugates, even with repeated infusions, only relatively low-grade toxicity of nausea and vomiting, or a controlled neutropenia, was observed. Another surprising result is the absence of accumulation of antibody-drug conjugates, unlike other products with SN-38 conjugated to albumin, PEG, or other carriers. The absence of accumulation even after repeated or increased dosing is associated with improved tolerability and absence of severe toxicity. These surprising results allow for optimization of dosage and delivery regimens with unexpectedly high efficiency and low toxicity. The claimed method provides 15% or more, preferably 20% or more, preferably 30% or more, more preferably 40% or more, shrinkage (as measured by longest diameter) of solid tumors in individuals with previously resistant cancer. One of ordinary skill will recognize that tumor size can be measured by a variety of different techniques, such as total tumor volume, maximum tumor size in any dimension, or a combination of size measurements in several dimensions. This may be done using standard radiological procedures such as computed tomography, ultrasonography, and/or positron emission tomography. The method of sizing is less important than the tendency of tumor size reduction observed by immunoconjugate treatment, preferably resulting in elimination of the tumor.

While the immunoconjugate may be administered as a regular bolus, in an alternative embodiment, the immunoconjugate may be administered by continuous infusion of the antibody-drug conjugate. To increase Cmax and prolong PK of the immunoconjugate in blood, continuous infusion may be performed, for example, through an indwelling catheter. Such devices are known in the art, e.g.

Or PORT-A-

Catheters (see, e.g., Skolnik et al, the Drug monitor 32:741-48,2010), and any such known indwelling catheter may be used. A variety of continuous infusion pumps are also known in the art, and any such known infusion pump may be used. More preferably, these immunoconjugates can be administered by intravenous infusion over a relatively short period of 2 to 5 hours, more preferably 2-3 hours.

In particularly preferred embodiments, the immunoconjugate and dosing regimen may be effective in patients who are resistant to standard therapy. For example, the hL243-SN-38 immunoconjugate can be administered to a patient that does not respond to prior irinotecan (parent agent of SN-38) therapy. Surprisingly, irinotecan-resistant patients may exhibit partial or even complete response to hL 243-SN-38. The ability of the immunoconjugate to specifically target tumor tissue can overcome tumor resistance through improved targeting and enhanced delivery of therapeutic agents. Combinations of different SN-38 immunoconjugates or SN-38-antibody conjugates combined with antibodies conjugated to radionuclides, toxins, or other drugs can provide even greater efficacy and/or reduced toxicity.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present invention. Embodiments may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 shows the structure of IMMU-140(hL243-CL 2A-SN-38).

FIG. 2. comparable combinations of IMMU-114 and IMMU-140. Binding curves were obtained for SN-38 conjugated (IMMU-140) and naked (hL243 in IMMU-1140 form). Control non-specific antibody (h679) showed no binding to HLA-DR + cells.

FIG. 3 in vivo efficacy of IMMU-140 versus IMMU-114 in MOLM-14AML xenografts.

FIG. 4 in vivo efficacy of IMMU-140 versus IMMU-114 in MN-60ALL xenografts.

FIG. 5 in vivo efficacy of IMMU-140 versus IMMU-114 in CAG MM xenografts.

FIG. 6 in vivo efficacy of IMMU-140 versus IMMU-114 in JVM-3CLL xenografts.

FIG. 7 binding of hL243- γ 4P to human melanoma cells in vitro.

Figure 8 efficacy of IMMU-140 in human melanoma xenografts in vivo.

Detailed Description

Definition of

In the following description, a number of terms are used, and the following definitions are provided to facilitate understanding of the claimed subject matter. Terms not explicitly defined herein are used according to their ordinary and customary meaning.

Unless otherwise stated, all references to "a", "an", and "the" are intended to mean that the elements are not in any way limitingOne (a)OrOne (an)Meaning "one or more".

Term "About"is used herein to denote plus or minus ten percent (10%) of a value. For example, "about 100" refers to any number between 90 and 110.

As used herein, the term "a" or "an" refers to a compound,antibodiesRefers to a full-length (i.e., naturally occurring or formed by the process of recombination of normal immunoglobulin gene fragments) immunoglobulin molecule (e.g., an IgG antibody) or an antigen-binding portion of an immunoglobulin molecule, e.g., an antibody fragment. Antibodies or antibody fragments may be conjugated or otherwise derivatized within the scope of the claimed subject matter. Such antibodies include, but are not limited to, IgG1, IgG2, IgG3, IgG4 (and IgG4 subtypes), and IgA isotypes. As used below, the abbreviation "MAb" is used interchangeably to refer to an antibody, antibody fragment, monoclonal antibody, or multispecific antibody.

Antibody fragmentsIs part of an antibody, e.g. F (ab') ₂ 、F(ab) ₂ Fab', Fab, Fv, scFv (single chain Fv), single domain antibody (DAB or VHH) and analogs, including the above-referenced half-molecule of IgG4 (van der Neut Kolfschoen et al (Science)2007; 317(14 Sept): 1554-1557). Regardless of structure, the antibody fragment used binds to the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes synthetic or genetically engineered proteins that function as antibodies by binding to a particular antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv" fragments consisting of the variable regions of the heavy and light chains, as well as recombinant single chain polypeptide molecules in which the light and heavy variable regions are joined by a peptide linker ("scFv proteins"). Fragments can be constructed in different ways to produce multivalent and/or multispecific binding forms.

Naked antibodyTypically an intact antibody that is not conjugated to a therapeutic agent. Naked antibodies may exhibit therapeutic and/or cytotoxic effects, for example through Fc-dependent functions such as complement fixation (CDC) and ADCC (antibody-dependent cellular cytotoxicity). However, other mechanisms, such as apoptosis, anti-angiogenesis, anti-metastatic activity, anti-adhesion activity, inhibition of heterotypic or homotypic adhesion, and interference with signaling pathways may also provide therapeutic effects. Naked antibodies include polyclonal and monoclonal antibodies, naturally occurring antibodies or recombinant antibodies, such as chimeric, humanized or human antibodies and fragments thereof. In some cases, "naked antibody" may also refer to "naked" antibody fragments. As defined herein, "naked" is synonymous with "unconjugated" and means not linked to or conjugated to a therapeutic agent.

Chimeric antibodiesIs a recombinant protein comprising variable domains of both the heavy and light chains comprising Complementarity Determining Regions (CDRs) of an antibody from one species, preferably a rodent antibody, more preferably a murine antibody, while the constant domains of the antibody molecule are from the constant domains of a human antibody.

Humanized antibodiesAre recombinant proteins in which the CDRs of an antibody from one species (e.g., a murine antibody) are transferred from the heavy and light chain variable chains of a murine antibody to human heavy and light variable domains (framework regions). The constant domains of the antibody molecule are derived from the constant domains of human antibodies. In some cases, specific residues of the framework regions of the humanized antibody are contacted, particularly with the CDR sequencesOr those residues in proximity thereto, may be modified, for example by replacement with the corresponding residues from a murine, rodent, human-like primate or other antibody of origin.

Human antibodiesAre, for example, antibodies obtained from transgenic mice that have been "engineered" to produce human antibodies in response to antigen challenge. In this technique, elements of the human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for various antigens, and mice can be used to produce secreted human antibody hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet.7:13(1994), Lonberg et al, Nature 368:856(1994), and Taylor et al, int. Immun.6:579 (1994). Fully human antibodies can also be constructed by genetic or chromosomal transfection methods as well as phage display techniques, all of which are known in the art. See, e.g., McCafferty et al, Nature 348:552-553(1990) for the in vitro production of human antibodies and fragments thereof from immunoglobulin variable domain gene banks from non-immunized donors. In this technique, human antibody variable domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of genes encoding antibodies exhibiting these properties. In this way, the phage mimics some of the characteristics of the B cell. Phage display can be performed in a variety of formats, for a review of these see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993). Human antibodies can also be produced by activated B cells in vitro. See U.S. Pat. nos. 5,567,610 and 5,229,275, the respective examples of which are incorporated herein by reference in their entirety.

Therapeutic agentsAre atoms, molecules or compounds that are useful in the treatment of disease. Examples of therapeutic agents include, but are not limited to, antibodies, antibody fragments, immunoconjugates, antibodies, immunoconjugates, and the like,Drugs, cytotoxic agents, pro-apoptotic agents, toxins, nucleases (including dnazymes and rnases), hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes, radionuclides, oligonucleotides, interfering RNAs, sirnas, RNAi, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins or peptides, or combinations thereof.

ImmunoconjugatesIs an antibody, antigen-binding antibody fragment, antibody complex, or antibody fusion protein conjugated to a therapeutic agent. Conjugation may be covalent or non-covalent. Preferably, the conjugation is covalent.

As used herein, the termAntibody fusion proteinsAre recombinantly produced antigen-binding molecules in which one or more natural antibodies, single chain antibodies or antibody fragments are linked to another moiety such as a protein or peptide, toxin, cytokine, hormone, or the like. In certain preferred embodiments, a fusion protein may comprise two or more identical or different antibodies, antibody fragments, or single chain antibodies fused together, which may bind to the same epitope, different epitopes on the same antigen or different antigens.

ImmunomodulatorIs a therapeutic agent that, when present, alters, inhibits or stimulates the body's immune system. Typically, the immunomodulator used stimulates immune cells to proliferate or activate in an immune response cascade, such as macrophages, dendritic cells, B cells and/or T cells. However, in some cases, the immunomodulator may inhibit proliferation or activation of immune cells. An example of an immunomodulator as described herein is a cytokine, which is a soluble small protein of about 5-20kDa, one cell population of which (e.g., primed T-lymphocytes) is released upon contact with a specific antigen, and acts as an intercellular mediator between cells. As the skilled person will appreciate, examples of cytokines include lymphokines, monokines, interleukins and a variety of related signaling molecules, such as Tumor Necrosis Factor (TNF) and interferons. Chemokines are a subset of cytokines. Certain interleukins and interferons are examples of cytokines that stimulate the proliferation of T cells or other immune cells. Exemplary ofInterferons include interferon- α, interferon- β, interferon- γ, and interferon- λ.

CPTIs an abbreviation for camptothecin, and as used herein, CPT means camptothecin itself or an analog or derivative of camptothecin, such as SN-38. The structures of camptothecin and some of its analogs (rings with numbers and labels indicated by letters a-E) are shown in formula 1 below and in panel 1.

FIG. 1 shows a schematic view of a

Camptothecin conjugates

Non-limiting methods and compositions for preparing immunoconjugates comprising a camptothecin therapeutic agent linked to an antibody or antigen-binding antibody fragment are described below. In a preferred embodiment, the solubility of the drug is enhanced by placing a defined polyethylene glycol (PEG) moiety (i.e., a PEG containing a defined number of monomeric units) between the drug and the antibody, wherein the defined PEG is a low molecular weight PEG, preferably containing 1-30 monomeric units, more preferably containing 1-12 monomeric units, and most preferably containing 7-8 monomeric units.

Preferably, the first linker is attached to the drug at one end and may be terminated with an acetylene or azide group at the other end. The first linker may comprise a defined PEG moiety having an azido or ethynyl group at one end and a different reactive group, such as a carboxylic acid or hydroxyl group, at the other end. The bifunctional defined PEG may be attached to an amine group of an amino alcohol, the latter hydroxyl group may be attached to a hydroxyl group on the drug in the form of a carbonate. Alternatively, the non-azide (or acetylene) moiety of the defined bifunctional PEG is optionally attached to the N-terminus of an L-amino acid or polypeptide, whose C-terminus is attached to the amino group of an amino alcohol, and the hydroxyl group of the latter is attached to the hydroxyl group of the drug, respectively in the form of a carbonate or carbamate.

A second linker comprising an antibody coupling group and a reactive group complementary to the azide (or acetylene) group of the first linker (i.e., acetylene (or azide)) can be reacted with the drug- (first linker) conjugate by an acetylene-azide cycloaddition reaction to provide a final bifunctional drug product that can be used to bind to an antibody that targets a disease. The antibody coupling group is preferably a thiol or thiol-reactive group.

The following provides a method for the selective regeneration of 10-hydroxy in the presence of C-20 carbonate in a drug-linker precursor formulation involving a CPT analog such as SN-38. Other protecting groups for reactive hydroxyl groups in the drug (e.g., phenolic hydroxyl groups in SN-38) may also be used, such as t-butyldimethylsilyl or t-butyldiphenylsilyl, and deprotected by tetrabutylammonium fluoride prior to attachment of the derivatized drug to the antibody coupling moiety. Alternatively, instead of "BOC", the 10-hydroxyl group of the CPT analogue is protected as an ester or carbonate, allowing the bifunctional CPT to be conjugated to an antibody without prior deprotection of the protecting group. The protecting group is readily deprotected under physiological pH conditions after administration of the bioconjugate.

In acetylene-azide coupling (known as 'click chemistry'), the azide moiety may be on L2 and the acetylene moiety on L3. Alternatively, L2 may contain acetylene and L3 contains azide. 'click chemistry' refers to a copper (+1) -catalyzed cycloaddition reaction between an acetylene moiety and an azide moiety (Kolb HC and Sharpless KB, drug Discov Today 2003; 8:1128-37), although alternative forms of click chemistry are known and can be used. Click chemistry is performed in aqueous solution at near neutral pH conditions and is therefore suitable for drug conjugation. Click chemistry has the advantage that it is chemoselective and complements other well-known conjugation chemistries, such as thiol-maleimide reactions.

While the present application focuses on the use of antibodies or antibody fragments as targeting moieties, the skilled artisan will recognize that when the conjugate comprises an antibody or antibody fragment, another type of targeting moiety, such as an aptamer, avimer, affibody (affibody), or peptide ligand, may be substituted.

An exemplary preferred embodiment relates to conjugates of a drug derivative of general formula 2 and an antibody,

MAb-[L2]-[L1]-[AA] _m -[A’]-medicament (2)

Wherein the MAb is an antibody that targets a disease; l2 is a crosslinker component comprising an antibody coupling moiety and one or more acetylene (or azide) groups; l1 comprises a defined PEG having an azide (or acetylene) complementary to the acetylene (or azide) moiety in L2 at one end and a reactive group such as a carboxylic acid or hydroxyl group at the other end; AA is an L-amino acid; m is an integer having a value of 0,1, 2,3 or 4; a' is a further spacer selected from ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol or a substituted or unsubstituted ethylenediamine. The L amino acid of 'AA' is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. If the A' group contains a hydroxyl group, it is attached to the hydroxyl or amino group of the drug in the form of a carbonate or carbamate, respectively.

In a preferred embodiment of formula 2, A' is a substituted ethanolamine derived from an L-amino acid, wherein the carboxylic acid group of the amino acid is partially substituted with a hydroxymethyl group. A' may be derived from any of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

A preferred embodiment, designated MAb-CL2A-SN-38, is shown below.

In the case of camptothecin containing a 10-hydroxy group (e.g., SN-38), other embodiments are possible. In the example of SN-38 as a drug, the more reactive 10-hydroxy group of the drug is derivatized, while the 20-hydroxy group is unaffected. In formula 2, a' is a substituted ethylenediamine.

In another preferred embodiment, the L1 component of the conjugate contains a defined polyethylene glycol (PEG) spacer having 1-30 repeating monomer units. In a further preferred embodiment, the PEG is a defined PEG having 1-12 repeating monomer units. The introduction of PEG may involve the use of commercially available heterobifunctional PEG derivatives. The heterobifunctional PEG may contain an azide or acetylene group. Examples of heterobifunctional defined PEGs containing 8 repeating monomer units, where 'NHS' is a succinimidyl group, are given in formula 3 below:

in a preferred embodiment, L2 has a plurality of acetylene (or azide) groups in the range of 2 to 40, but preferably 2 to 20, more preferably 2 to 5, and a single antibody binding moiety.

Representative SN-38 conjugates of antibodies containing multiple drug molecules and a single antibody binding moiety are shown below. The 'L2' component of this structure is linked to 2 alkynyl groups resulting in the attachment of two azide-linked SN-38 molecules. The linkage to the MAb is denoted as succinimide.

Wherein the R residues are:

in some preferred embodiments, when the bifunctional drug contains a thiol-reactive moiety as an antibody binding group, a thiolating reagent is used to generate a thiol on the antibody at a lysine group of the antibody. Methods for introducing thiol groups into antibodies by modifying lysine groups of MAbs are well known in the art (Wong, Chemistry of protein conjugation and cross-linking, CRC Press, Inc., Boca Raton, FL (1991), pages 20-22). Alternatively, a slight reduction of the interchain disulfide bond on an antibody using a reducing agent such as Dithiothreitol (DTT) (Willner et al, Bioconjugate chem.4:521-527(1993)) can produce 7 to 10 thiols on the antibody; this has the advantage of introducing multiple drug moieties in the inter-chain regions of the MAb remote from the antigen binding region. In a more preferred embodiment, the attachment of SN-38 to a reduced disulfide thiol results in the formation of an antibody-SN-38 immunoconjugate in which 6 to 8 SN-38 moieties are covalently attached per antibody molecule. Other methods for providing cysteine residues for attachment of drugs or other therapeutic agents are known, such as the use of cysteine engineered antibodies (see U.S. Pat. No. 7,521,541, the examples section of which is incorporated herein by reference.)

In an alternative preferred embodiment, the chemotherapeutic moiety is selected from the group consisting of Doxorubicin (DOX), epirubicin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolinyl-doxorubicin (2-PDOX), Pro-2PDOX, CPT, 10-hydroxycamptothecin, SN-38, topotecan, lurtotecan (lurtotecan), 9-aminocamptothecin, 9-nitrocamptothecin, taxane, geldanamycin (geldanamycin), ansamycin (ansamycins), and epothilone (epothilones). In a more preferred embodiment, the chemotherapeutic moiety is SN-38. Preferably, in the conjugates of the preferred embodiments, the antibody is linked to at least one chemotherapeutic moiety; preferably 1 to about 12 chemotherapeutic moieties; more preferably from about 6 to about 12 chemotherapeutic moieties, and most preferably from about 6 to 8 chemotherapeutic moieties.

Furthermore, in some preferred embodiments, linker component 'L2' comprises a thiol group that reacts with a thiol-reactive residue introduced at one or more lysine side chain amino groups of the antibody. In this case, the antibody is pre-derivatized with a thiol-reactive group, such as maleimide, vinyl sulfone, bromoacetamide, or iodoacetamide, by procedures well described in the art.

In the context of this work, a process has surprisingly been found by which CPT drug linkers can be prepared, wherein CPT additionally has a 10-hydroxy group. The method includes, but is not limited to, protecting the 10-hydroxy group as a tert-Butoxycarbonyl (BOC) derivative, followed by preparation of the penultimate intermediate of the drug-linker conjugate. Typically, removal of the BOC group requires treatment with a strong acid such as trifluoroacetic acid (TFA). Under these conditions, the CPT 20-O-linker carbonate containing the protecting group to be removed is also susceptible to cleavage, thereby producing unmodified CPT. Indeed, as demonstrated in the art, the rationale behind the use of a mild removable methoxytrityl (MMT) protecting group for the lysine side chains of the linker molecule is just to avoid this possibility (Walker et al, 2002). It was found that the phenolic BOC protecting group can be selectively removed by carrying out the reaction for a short time, optimally 3 to 5 minutes. Under these conditions, the main product is ` BOC ` with the 10-hydroxyl position removed, while the carbonate in the ` 20 ` position is intact.

Another approach involves protecting the 10-hydroxyl position of the CPT analog with a group other than "BOC" so that the final product is ready for coupling to an antibody without the need to deprotect the 10-OH protecting group. The 10-hydroxy protecting group that converts 10-OH to a phenolic carbonate or ester is readily deprotected after administration of the conjugate in vivo, either at physiological pH or by esterase. He et al have described that phenolic carbonates in the 10 position of 10-hydroxycamptothecin are removed more rapidly than versatates in the 20 position under physiological conditions (He et al, Bioorganic)&Medicinal Chemistry 12:4003-4008 (2004)). The 10-hydroxy protecting group on SN-38 can be 'COR', where R can be a substituted alkyl, such as "N (CH) ₃ ) ₂ -(CH ₂ ) _n - ", where n is 1 to 10 and where the terminal amino group is optionally in the form of a quaternary salt for enhanced water solubility, or a simple alkyl residue, e.g." CH ₃ -(CH ₂ ) _n - ", where n is 0 to 10, or it may be an alkoxy moiety, for example" CH ₃ -(CH ₂ ) N-O- "wherein N is 0-10, or" N (CH) ₃ ) ₂ -(CH ₂ ) _n -O- ", wherein n is 2-10, or" R "" ₁ O-(CH ₂ -CH ₂ -O) _n -CH ₂ -CH ₂ -O- ", wherein R ₁ Is ethyl or methylAnd n is an integer having a value of 0 to 10. If the final derivative is a carbonate, these 10-hydroxy derivatives can be readily prepared by treatment with the chloroformate of the selected reagent. Typically, a 10-hydroxy group containing camptothecin, such as SN-38, is treated with a molar equivalent of chloroformate in dimethylformamide using triethylamine as the base. Under these conditions, the 20-OH position is not affected. To form the 10-O-ester, the acid chloride of the selected reagent is used.

In a preferred method of preparing a conjugate of a drug derivative and an antibody of formula 2, wherein the descriptors L2, L1, AA and A-X are as described in the previous section, a bifunctional drug moiety [ L2 ] is first prepared]-[L1]-[AA] _m -[A-X]-a drug, followed by conjugation of a bifunctional drug moiety to an antibody (denoted herein as "MAb").

In a preferred method of preparing a conjugate of a drug derivative and an antibody of formula 2, wherein the descriptors L2, L1, AA and a-OH are as described in the previous section, the bifunctional drug moiety is prepared by first linking the a-OH to the C-terminus of AA via an amide bond, and then coupling the amine terminus of AA to the carboxylic acid group of L1. If AA is not present (i.e., m is 0), then a-OH is directly attached to L1 through an amide bond. Cross-linked linker [ L1]-[AA] _m -[A-OH]Attached to the hydroxy or amino group of the drug, which is then attached to the L1 moiety by click chemistry reaction between the azide (or acetylene) and acetylene (or azide) groups in L1 and L2.

In one embodiment, the antibody is a monoclonal antibody (MAb). In other embodiments, the antibody may be a multivalent and/or multispecific MAb. The antibody may be a murine, chimeric, humanized or human monoclonal antibody, and the antibody may be intact, fragment (Fab, Fab', F (ab)) ₂ 、F(ab’) ₂ ) Or in subfragment (single chain construct) form, or as IgG1, IgG2a, IgG3, IgG4, IgA isotype or a sub-molecule thereof.

In a preferred embodiment, the antibody binds to an antigen or epitope of an antigen expressed on a cancer or malignant cell. The cancer cell is preferably a cell from a hematopoietic tumor, carcinoma, sarcoma, melanoma, or glioma. Preferred malignant tumors to be treated according to the present invention are malignant solid tumors or hematopoietic tumors.

In a preferred embodiment, the intracellular cleavable moiety may be cleaved, and in particular by esterases and peptidases, after internalization into the cell by binding of the MAb-drug conjugate to its receptor.

anti-HLA-DR antibodies

In a preferred embodiment, the immunoconjugate comprises an anti-HLA-DR antibody, such as a humanized L243 antibody. The humanized L243 antibody binds to the same epitope on HLA-DR as the parent murine L243 antibody, but has reduced immunogenicity. mL243 is a monoclonal antibody previously described by Lampson and Levy (J Immunol,1980,125:293), which has been deposited with the American Type Culture Collection, Rockville, Md. under accession number ATCC HB 55.

The humanized L243 antibody comprises the L243 heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:1), CDR2(WINTYTREPTYADDFKG, SEQ ID NO:2) and CDR3(DITAVVPTGFDY, SEQ ID NO:3) and the light chain CDR1(RASENIYSNLA, SEQ ID NO:4), CDR2(AASNLAD, SEQ ID NO:5) and CDR3(QHFWTTPWA, SEQ ID NO:6) linked to the human antibody FR and constant region sequences. In a more preferred embodiment, one or more murine FR amino acid residues are replaced by the corresponding human FR residues, particularly at positions adjacent or near the CDR residues. Exemplary murine V that can be substituted in humanized design _H Residues at the following positions: f27, K38, K46, a68 and F91. Exemplary murine V that can be substituted in humanized design _L Residues at the following positions: r37, K39, V48, F49 and G1.

A particularly preferred format of the hL243 antibody, which comprises FR sequences from human RF-TS3, NEWM and REI antibodies, is described in U.S. patent No. 7,612,180 (which is incorporated herein by reference). However, in other embodiments, the FR residues may be from any suitable human immunoglobulin, provided that the humanized antibody can fold such that it retains the ability to specifically bind to HLA-DR. Preferably, the type of human Framework (FR) used is of the same/similar class/type as the donor antibody. More preferably, the human FR sequence is selected to have a high degree of sequence homology with the corresponding murine FR sequence, particularly in the nullOr a position adjacent or near the CDR. According to this embodiment, humanized L243V _H Or V _L The framework of (i.e., FR1-4) can be derived from a combination of human antibodies. Examples of human frameworks that can be used to construct CDR-grafted humanized antibodies are LAY, POM, TUR, TEI, KOL, NEWM, REI, RF and EU. Preferably, human RF-TS3FR1-3 and NEWM FR4 are used for the heavy chain and REI FR1-4 is used for the light chain. The variable domain residue numbering system used herein is described in: kabat et al, (1991), Sequences of Proteins of Immunological Interest, 5 th edition, United States Department of Health and Human Services

The light and heavy chain variable domains of the humanized antibody molecule may be fused to human light or heavy chain constant domains. Human constant domains can be selected for the proposed antibody function. In one embodiment, the human constant domain may be selected based on a lack of effector function. The heavy chain constant domain fused to the heavy chain variable region may be those of human IgA (α 1 or α 2 chain), IgG (γ 1, γ 2, γ 3 or γ 4 chain) or IgM (μ chain). Light chain constant domains that can be fused to the light chain variable region include human λ and κ chains.

In one embodiment of the invention, γ l chains are used. In yet another specific embodiment, a γ 4 chain is used. In certain instances, use of γ 4 chains may increase tolerance to hL243 in a subject (reduce side effects and infusion reactions, etc.).

In one embodiment, analogs of the human constant domain may be used. These include, but are not limited to, those constant domains that contain one or more additional amino acids compared to the corresponding human domain or those constant domains in which one or more existing amino acids of the corresponding human domain have been deleted or altered. These domains can be obtained, for example, by oligonucleotide-directed mutagenesis.

In a specific embodiment, the anti-HLA-DR antibody or fragment thereof may be a fusion protein. The fusion protein may comprise one or more anti-HLA-DR antibodies or fragments thereof. In various embodiments, the fusion protein may also comprise one or more additional antibodies to different antigens, or may comprise different effector proteins or peptides, such as cytokines. For example, the different antigen may be a tumor marker selected from a B cell lineage antigen (e.g., CD19, CD20, or CD22) for use in the treatment of a B cell malignancy. In another example, different antigens may be expressed on other cells that cause other types of malignancies. Furthermore, the cell marker may be a non-B cell lineage antigen, for example selected from HLA-DR, CD3, CD33, CD52, CD66, MUC1 and TAC.

In one embodiment, the anti-HLA-DR antibody can be combined with other antibodies and used to treat a subject having or suspected of having a disease. According to this embodiment, the anti-HLA-DR antibody or fragment thereof can be combined with an anti-cancer monoclonal antibody, such as a humanized monoclonal antibody (e.g., hA20, anti-CD 20Mab) and used to treat cancer. It is contemplated herein that anti-HLA-DR antibodies can be used as a separate antibody composition in combination with one or more other separate antibody compositions, or as bifunctional antibodies containing, for example, one anti-HLA-DR and one other anti-tumor antibody (e.g., hA 20). In another embodiment, the antibody may target a B cell malignancy. B cell malignancies can consist of indolent forms of B cell lymphoma, aggressive forms of B cell lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, waldenstrom's macroglobulinemia, and multiple myeloma. Other non-malignant B cell disorders and related diseases that can be treated with the antibodies of the invention include a number of autoimmune and immune dysregulated diseases, such as sepsis and septic shock.

General antibody technology

Techniques for making monoclonal antibodies against virtually any target antigen are well known in the art. See, for example,

and Milstein, Nature 256:495(1975), and Coligan et al, (ed.), Current PROTOCOLS IN IMMUNOLOGY, Vol.1, pp.2.5.1-2.6.7 (John Wiley&Sons 1991). Briefly, monoclonal antibodies can be obtained by: injecting a composition containing a human antigen (e.g., human HLA-DR) into a mouse, removing spleen to obtain B lymphocytes, and fusing the B lymphocytes with myeloma cellsTo produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to human antigens, culturing the clones that produce antibodies to the antigens, and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such separation techniques include affinity chromatography with protein-A or protein-G sepharose, size exclusion chromatography, and ion exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. See also Baines et al, "Purification of Immunoglobulin G (IgG)," METHODS IN MOLECULAR BIOLOGY, Vol.10, pp.79-104 (The Humana Press, Inc.1992).

After the initial production of antibodies to the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below.

One skilled in the art will recognize that the claimed methods and compositions may use any of a variety of antibodies known in the art. The antibodies used are commercially available from a variety of known sources. For example, a variety of antibody secreting hybridoma lines are available from the american type culture collection (ATCC, Manassas, VA). A number of antibodies against a variety of disease targets, including but not limited to tumor associated antigens, have been deposited with the ATCC and/or have disclosed variable region sequences and are useful in the claimed methods and compositions. See, for example, the following U.S. patent nos.: 7,312,318, respectively; 7,282,567, respectively; 7,151,164, respectively; 7,074,403, respectively; 7,060,802, respectively; 7,056,509, respectively; 7,049,060, respectively; 7,045,132, respectively; 7,041,803, respectively; 7,041,802, respectively; 7,041,293, respectively; 7,038,018, respectively; 7,037,498, respectively; 7,012,133; 7,001,598, respectively; 6,998,468, respectively; 6,994,976, respectively; 6,994,852, respectively; 6,989,241, respectively; 6,974,863, respectively; 6,965,018, respectively; 6,964,854, respectively; 6,962,981, respectively; 6,962,813, respectively; 6,956,107, respectively; 6,951,924, respectively; 6,949,244, respectively; 6,946,129, respectively; 6,943,020, respectively; 6,939,547, respectively; 6,921,645, respectively; 6,921,645, respectively; 6,921,533, respectively; 6,919,433, respectively; 6,919,078, respectively; 6,916,475, respectively; 6,905,681, respectively; 6,899,879, respectively; 6,893,625; 6,887,468; 6,887,466, respectively; 6,884,594, respectively; 6,881,405, respectively; 6,878,812, respectively; 6,875,580, respectively; 6,872,568, respectively; 6,867,006, respectively; 6,864,062, respectively; 6,861,511, respectively; 6,861,227, respectively; 6,861,226, respectively; 6,838,282, respectively; 6,835,549, respectively; 6,835,370, respectively; 6,824,780, respectively; 6,824,778, respectively; 6,812,206, respectively; 6,793,924, respectively; 6,783,758, respectively; 6,770,450, respectively; 6,767,711, respectively; 6,764,688, respectively; 6,764,681, respectively; 6,764,679, respectively; 6,743,898, respectively; 6,733,981, respectively; 6,730,307, respectively; 6,720,155, respectively; 6,716,966, respectively; 6,709,653, respectively; 6,693,176; 6,692,908, respectively; 6,689,607, respectively; 6,689,362; 6,689,355, respectively; 6,682,737, respectively; 6,682,736; 6,682,734, respectively; 6,673,344, respectively; 6,653,104, respectively; 6,652,852, respectively; 6,635,482; 6,630,144, respectively; 6,610,833, respectively; 6,610,294, respectively; 6,605,441, respectively; 6,605,279, respectively; 6,596,852, respectively; 6,592,868, respectively; 6,576,745, respectively; 6,572,856, respectively; 6,566,076, respectively; 6,562,618, respectively; 6,545,130, respectively; 6,544,749, respectively; 6,534,058, respectively; 6,528,625, respectively; 6,528,269, respectively; 6,521,227, respectively; 6,518,404, respectively; 6,511,665; 6,491,915, respectively; 6,488,930, respectively; 6,482,598; 6,482,408, respectively; 6,479,247, respectively; 6,468,531, respectively; 6,468,529, respectively; 6,465,173; 6,461,823, respectively; 6,458,356, respectively; 6,455,044, respectively; 6,455,040, 6,451,310; 6,444,206' 6,441,143; 6,432,404, respectively; 6,432,402, respectively; 6,419,928, respectively; 6,413,726, respectively; 6,406,694, respectively; 6,403,770, respectively; 6,403,091, respectively; 6,395,276, respectively; 6,395,274, respectively; 6,387,350, respectively; 6,383,759, respectively; 6,383,484, respectively; 6,376,654, respectively; 6,372,215, respectively; 6,359,126, respectively; 6,355,481, respectively; 6,355,444, respectively; 6,355,245, respectively; 6,355,244; 6,346,246, respectively; 6,344,198, respectively; 6,340,571, respectively; 6,340,459, respectively; 6,331,175, respectively; 6,306,393, respectively; 6,254,868, respectively; 6,187,287; 6,183,744, respectively; 6,129,914, respectively; 6,120,767; 6,096,289, respectively; 6,077,499; 5,922,302, respectively; 5,874,540; 5,814,440; 5,798,229, respectively; 5,789,554; 5,776,456; 5,736,119, respectively; 5,716,595, respectively; 5,677,136, respectively; 5,587,459, respectively; 5,443,953, 5,525,338, examples of each of which are incorporated herein in part by reference. These are merely exemplary, and a variety of other antibodies and hybridomas thereof are known in the art. The skilled artisan will recognize that antibody sequences or antibody-secreting hybridomas to virtually any disease-associated antigen can be obtained by simply searching the ATCC, NCBI, and/or USPTO databases for antibodies to the selected disease-associated target of interest. The antigen binding domain of the cloned antibody can be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production using standard techniques well known in the art. The isolated antibody can be conjugated to a therapeutic agent, such as camptothecin, using the techniques disclosed herein.

Chimeric and humanized antibodies

Chimeric antibodies are recombinant proteins in which the variable regions of a human antibody have been replaced by the variable regions of a mouse antibody, e.g., comprising the Complementarity Determining Regions (CDRs) of a mouse antibody. When administered to a subject, the chimeric antibodies exhibit reduced immunogenicity and increased stability. Methods for constructing chimeric antibodies are well known in the art (e.g., Leung et al, 1994, Hybridoma 13: 469).

Chimeric monoclonal antibodies can be humanized by transferring mouse CDRs from the heavy and light chain variable chains of a mouse immunoglobulin into the corresponding variable domains of a human antibody. The mouse Framework Region (FR) in the chimeric monoclonal antibody is also replaced by human FR sequences. To maintain the stability and antigen specificity of the humanized monoclonal, one or more human FR residues may be replaced with mouse counterpart residues. The humanized monoclonal antibodies are useful for therapeutic treatment of a subject. Techniques for generating humanized monoclonal antibodies are well known in the art. (see, e.g., Jones et al, 1986, Nature,321: 522; Riechmann et al, Nature,1988,332: 323; Verhoeyen et al, 1988, Science,239: 1534; Carter et al, 1992, Proc.Nat' l Acad.Sci.USA,89: 4285; Sandhu, Crit.Rev.Biotech.,1992,12: 437; Tempest et al, 1991, Biotechnology 9: 266; Singer et al, J.Immun.,1993,150:2844.)

Other embodiments may relate to non-human primate antibodies. General techniques for generating therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al, WO 91/11465(1991), and Losman et al, int.J. cancer46:310 (1990). In another embodiment, the antibody can be a human monoclonal antibody. Such antibodies can be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigen challenge, as described below.

Human antibodies

Methods for producing fully human antibodies using combinatorial approaches or transgenic animals transformed with human immunoglobulin loci are known in the art (e.g., Mancini et al, 2004, New Microbiol.27: 315-28; Conrad and Scheller,2005, comb. chem. high through Screen.8: 117-26; Brekke and Loset,2003, curr. Opin. Phamacol.3: 544-50; each incorporated herein by reference). Such fully human antibodies are expected to exhibit even fewer side effects than chimeric or humanized antibodies and to function as substantially endogenous human antibodies in vivo. In certain embodiments, the claimed methods and procedures may utilize human antibodies produced by these techniques.

In one alternative, phage display technology can be used to produce human antibodies (e.g., Dantas-Barbosa et al, 2005, Genet. mol. Res.4:126-40, incorporated herein by reference). Human antibodies can be produced by normal humans or by humans exhibiting particular disease states (e.g., cancer) (Dantas-Barbosa et al, 2005). An advantage of constructing human antibodies from diseased individuals is that the circulating antibody repertoire may be biased towards antibodies directed against disease-associated antigens.

In one non-limiting example of this approach, Dantas-Barbosa et al (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Typically, total RNA is obtained from circulating blood lymphocytes (supra) recombinant fabs are cloned from μ, γ, and κ chain antibody libraries and inserted into phage display libraries (supra) RNA is converted to cDNA and used to prepare Fab cDNA libraries using specific primers for heavy and light chain immunoglobulin sequences (Marks et al, 1991, j.mol.biol.222:581-97, incorporated herein by reference). Library construction was performed according to Andris-Widhopf et al (2000, phase Display Laboratory Manual, Barbas et al (ed.), 1 st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pages 9.1 to 9.22, incorporated herein by reference). The final Fab fragments were digested with restriction endonucleases and inserted into the phage genome to make phage display libraries. These libraries can be screened by standard phage display methods. The skilled artisan will recognize that this technique is merely exemplary, and that any known method of making and screening human antibodies or antibody fragments by phage display may be used.

In another alternative, transgenic animals that have been genetically engineered to produce human antibodies can be used to produce antibodies against essentially any immunogenic target using standard immunization protocols as described above. Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet.7:13(1994), Lonberg et al, Nature 368:856(1994), and Taylor et al, int.Immun.6:579 (1994). Non-limiting examples of such systems are from Abgenix (Fremont, CA)

(e.g., Green et al, 1999, J.Immunol. methods 231:11-23, incorporated herein by reference). In that

And similar animals, the mouse antibody genes have been inactivated and replaced with functional human antibody genes, while the rest of the mouse immune system remains intact.

Transformation with germline-configured YACs (Yeast Artificial chromosomes)

The YACs comprise a portion of the human IgH and Ig κ loci, including most of the variable region sequences, along with auxiliary gene and regulatory sequences. The repertoire of human variable regions can be used to generate antibody-producing B cells, which can be processed into hybridomas by known techniques. Immunising with target antigens

Human antibodies will be produced by a normal immune response, which can be harvested and/or produced by standard techniques described above. There are a plurality of

Strains were selected, each strain being capable of producing a different class of antibodies. Genetically produced human antibodies have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al, 1999). The skilled artisan will recognize that the claimed compositions and methods are not limited to use

The system, but can utilize any transgenic animal genetically engineered to produce human antibodies.

Production of antibody fragments

Some embodiments of the claimed methods and/or compositions may relate to antibody fragments. Such antibody fragments may be obtained, for example, by pepsin or papain digestion of intact antibodies by conventional methods. For example, antibody fragments can be generated by enzymatic cleavage of antibodies with pepsin to provide a peptide represented as F (ab') ₂ The 5S fragment of (1). The fragment can be further cleaved using a thiol reducing agent and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment. Exemplary methods for producing antibody fragments are disclosed below: U.S. Pat. nos. 4,036,945; U.S. Pat. nos. 4,331,647; nisonoff et al, 1960, arch.biochem.biophysis, 89: 230; porter,1959, biochem.j.,73: 119; edelman et al, 1967, METHODS IN ENZYMOLOGY, pp 422 (Academic Press), and Coligan et al, eds 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley&Sons)。

Other methods of cleaving antibodies can be used, such as isolating heavy chains to form monovalent light-heavy chain fragments, further cleaving fragments, and other enzymatic, chemical, or genetic techniques, so long as the fragments bind to the antigen recognized by the intact antibody. For example, the Fv fragment comprises V _H And V _L Association of chains. This association may be non-covalent, as described in Inbar et al, 1972, proc.nat' l.acad.sci.usa,69: 2659. Alternatively, the variable chains may be linked by intermolecular disulfide bonds or crosslinked by chemicals such as glutaraldehyde. See Sandhu,1992, Crit. Rev. Biotech, 12: 437.

Preferably, the Fv fragment comprises V linked by a peptide linker _H And V _L And (3) a chain. These single-chain antigen binding proteins (scFv) were prepared by constructing a structural gene comprising DNA sequences encoding V linked by an oligonucleotide linker sequence _H And V _L A domain. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, for example, E.coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging two V domains. Methods for producing scFv are well known in the art. See Whitlow et al, 1991, MethodsA company to Methods in Enzymology 2: 97; bird et al, 1988, Science,242: 423; U.S. Pat. nos. 4,946,778; pack et al, 1993, Bio/Technology,11:1271, and Sandhu,1992, crit. Rev. Biotech, 12: 437.

Another form of antibody fragment is a single domain antibody (dAb), sometimes referred to as a single chain antibody. Techniques for generating single domain antibodies are well known in the art (see, e.g., Cossins et al, Protein Expression and Purification,2007,51: 253-59; Shuntao et al, Molec Immunol 2006,43: 1912-19; Tanha et al, J.biol.chem.2001,276: 24774-. Other types of antibody fragments may comprise one or more Complementarity Determining Regions (CDRs). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDRs of an antibody of interest. Such genes are prepared, for example, by synthesizing the variable regions of RNA from antibody-producing cells using the polymerase chain reaction. See Larrick et al, 1991, Methods: A company to Methods in Enzymology 2: 106; ritter et al, (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, pp 166-179 (Cambridge University Press); birch et al (eds.), 1995, MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pp.137-185 (Wiley-Liss, Inc.)

Antibody variants

In certain embodiments, the antibody sequence (e.g., the Fc portion of an antibody) can be altered to optimize a physiological characteristic of the conjugate, such as half-life in serum. Methods for replacing amino acid sequences in proteins are well known in the art, e.g., by site-directed mutagenesis (e.g., Sambrook et al, Molecular Cloning, A laboratory manual, 2 nd edition, 1989). In preferred embodiments, the modifications may include the addition or removal of one or more glycosylation sites in the Fc sequence (e.g., U.S. patent No. 6,254,868, the examples section of which is incorporated herein by reference). In other preferred embodiments, specific amino acid substitutions in the Fc sequence can be made (e.g., Hornick et al, 2000, J Nucl Med 41: 355-62; Hinton et al, 2006, J Immunol 176: 346-56; Petkova et al, 2006, Int Immunol 18: 1759-69; U.S. Pat. No. 7,217,797; each of which is incorporated herein by reference).

Known antibodies

The antibodies used are commercially available from a variety of known sources. For example, a variety of antibody-secreting hybridoma lines are available from the american type culture collection (ATCC, Manassas, VA). A number of antibodies against a variety of disease targets, including but not limited to tumor associated antigens, have been deposited with the ATCC and/or have disclosed variable region sequences and are useful in the claimed methods and compositions. See, for example, the following U.S. patent nos.: 7,312,318, respectively; 7,282,567, respectively; 7,151,164, respectively; 7,074,403, respectively; 7,060,802, respectively; 7,056,509, respectively; 7,049,060, respectively; 7,045,132, respectively; 7,041,803; 7,041,802, respectively; 7,041,293, respectively; 7,038,018, respectively; 7,037,498; 7,012,133, respectively; 7,001,598, respectively; 6,998,468; 6,994,976, respectively; 6,994,852, respectively; 6,989,241, respectively; 6,974,863, respectively; 6,965,018, respectively; 6,964,854, respectively; 6,962,981; 6,962,813, respectively; 6,956,107; 6,951,924; 6,949,244, respectively; 6,946,129, respectively; 6,943,020, respectively; 6,939,547, respectively; 6,921,645, respectively; 6,921,645, respectively; 6,921,533, respectively; 6,919,433, respectively; 6,919,078, respectively; 6,916,475, respectively; 6,905,681, respectively; 6,899,879, respectively; 6,893,625, respectively; 6,887,468, respectively; 6,887,466, respectively; 6,884,594, respectively; 6,881,405, respectively; 6,878,812, respectively; 6,875,580, respectively; 6,872,568, respectively; 6,867,006, respectively; 6,864,062, respectively; 6,861,511, respectively; 6,861,227, respectively; 6,861,226, respectively; 6,838,282, respectively; 6,835,549, respectively; 6,835,370, respectively; 6,824,780, respectively; 6,824,778, respectively; 6,812,206, respectively; 6,793,924, respectively; 6,783,758; 6,770,450; 6,767,711, respectively; 6,764,688; 6,764,681, respectively; 6,764,679, respectively; 6,743,898, respectively; 6,733,981, respectively; 6,730,307, respectively; 6,720,155, respectively; 6,716,966; 6,709,653, respectively; 6,693,176, respectively; 6,692,908, respectively; 6,689,607, respectively; 6,689,362, respectively; 6,689,355, respectively; 6,682,737, respectively; 6,682,736; 6,682,734, respectively; 6,673,344, respectively; 6,653,104; 6,652,852, respectively; 6,635,482, respectively; 6,630,144, respectively; 6,610,833, respectively; 6,610,294, respectively; 6,605,441, respectively; 6,605,279, respectively; 6,596,852, respectively; 6,592,868, respectively; 6,576,745, respectively; 6,572, respectively; 856; 6,566,076, respectively; 6,562,618, respectively; 6,545,130, respectively; 6,544,749, respectively; 6,534,058, respectively; 6,528,625, respectively; 6,528,269, respectively; 6,521,227, respectively; 6,518,404, respectively; 6,511,665; 6,491,915, respectively; 6,488,930, respectively; 6,482,598, respectively; 6,482,408, respectively; 6,479,247, respectively; 6,468,531, respectively; 6,468,529, respectively; 6,465,173, respectively; 6,461,823, respectively; 6,458,356; 6,455,044, respectively; 6,455,040, respectively; 6,451,310, respectively; 6,444,206, respectively; 6,441,143, respectively; 6,432,404, respectively; 6,432,402, respectively; 6,419,928, respectively; 6,413,726, respectively; 6,406,694, respectively; 6,403,770, respectively; 6,403,091, respectively; 6,395,276, respectively; 6,395,274, respectively; 6,387,350, respectively; 6,383,759, respectively; 6,383,484, respectively; 6,376,654, respectively; 6,372,215, respectively; 6,359,126, respectively; 6,355,481, respectively; 6,355,444, respectively; 6,355,245, respectively; 6,355,244, respectively; 6,346,246, respectively; 6,344,198, respectively; 6,340,571, respectively; 6,340,459, respectively; 6,331,175, respectively; 6,306,393, respectively; 6,254,868, respectively; 6,187,287; 6,183,744, respectively; 6,129,914, respectively; 6,120,767, respectively; 6,096,289, respectively; 6,077,499; 5,922,302, respectively; 5,874,540; 5,814,440, respectively; 5,798,229, respectively; 5,789,554, respectively; 5,776,456; 5,736,119; 5,716,595, respectively; 5,677,136, respectively; 5,587,459; 5,443,953,5,525,338.

These are merely exemplary, and a variety of other antibodies and hybridomas thereof are known in the art. The skilled artisan will recognize that antibody sequences or antibody-secreting hybridomas to virtually any disease-associated antigen can be obtained by simply searching the ATCC, NCBI, and/or USPTO databases for antibodies to the selected disease-associated target of interest. The antigen binding domain of the cloned antibody can be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production using standard techniques well known in the art.

Exemplary antibodies that can be used include, but are not limited to, hR1 (anti-IGF-1R, U.S. Pat. No. 9,441,043), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20 (anti-CD 20, U.S. Pat. No. 7,151,164), hA19 (anti-CD 19, U.S. Pat. No. 7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1 (anti-CD 74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD 22, U.S. Pat. No. 5,789,554), hMu-9 (anti-CSAP, U.S. Pat. No. 7,387,772), hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM 5, U.S. Pat. No. 6,676,924), hMN-15 (anti-CEACAM 6, U.S. Pat. No. 8,287,865), hRS7 (anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM 6, U.S. Pat. No. 7,541,440), Ab124, and Ab125 (anti-CXCR 4, U.S. Pat. No. 7,138,496), the examples of each referenced patent or application being incorporated herein in part by reference. More preferably, the antibody is IMMU-31 (anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti-CEACAM 5), hMN-3 (anti-CEACAM 6), hMN-15 (anti-CEACAM 6), hLL1 (anti-CD 74), hLL2 (anti-CD 22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD 19) or hA20 (anti-CD 20). As used herein, the terms epratuzumab and hLL2 are interchangeable, as are the terms veltuzumab and hA20, and the terms hL243g4P, hL243 γ 4P and IMMU-114.

Alternative antibodies used include, but are not limited to, abciximab (anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD 52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD 33), ibritumomab (anti-CD 20), panitumumab (anti-EGFR), rituximab (anti-CD 20), tositumomab (anti-CD 20), trastuzumab (anti-ErbB 2), pembrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), abacuzumab (anti-CA-125), alemtuzumab (anti-EpCAM), alemtuzumab (anti-IL-6 receptor), benralizumab (anti-CD 125), obizumab (GA101, anti-CD 20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, us patent application 11/983,372, accession numbers ATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575), tosublizumab (anti-IL-6 receptor), basiliximab (anti-CD 25), dallizumab (anti-CD 25), efletuzumab (anti-CD 11a), GA101 (anti-CD 20; glycart Roche), natalizumab (anti- α 4 integrin), omalizumab (anti-IgE); anti-TNF-alpha antibodies, such as CDP571(Ofei et al, 2011, Diabetes 45:881-85), MTNFAI, M2TNFAI, M3 TNFAAI, M3TNFABI, M302B, M303(Thermo Scientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.), certolizumab (cetuzumab) (UCB, Brussels, Belgium), anti-CD 40L (UCB, Brussels, Belgium), adalimumab (Abbott, Abbott Park, Ill) and belimumab (Human Genome Sciences).

Comprehensive analysis of suitable antigen (or CD) targets on hematopoietic malignancies revealed by flow cytometry, which can be used as a guide to select suitable antibodies for drug-conjugated immunotherapy, is Craig and fonn, Blood prepublished online jan.15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigen consists of five different glycoproteins with similar structures, CD66a-e, which are encoded by the carcinoembryonic antigen (CEA) gene family members BCG, CGM6, NCA, CGM1, and CEA, respectively. These CD66 antigens (e.g., CEACAM6) are expressed primarily in granulocytes, normal epithelial cells of the gut, and tumor cells of various tissues. Also included as suitable targets for Cancer are Cancer testis antigens such as NY-ESO-1 (Theurilat et al, int.J.cancer 2007; 120(11):2411-7), and CD79a in myeloid leukemia (Kozlov et al, Cancer Genet. cytogene.2005; 163(l):62-7) and CD79B in B cell disease and non-Hodgkin's lymphoma (Poison et al, Blood 110(2): 616-. Many of the above antigens are disclosed in U.S. provisional application Ser. No. 60/426,379 entitled "Use of Multi-specific, Non-covalent Compounds for Targeted Delivery of Therapeutics", filed on 11/15/2002. Cancer stem cells are described as a more therapeutically resistant population of precursor malignant cells (Hill and Penis, J.Natl. Cancer Inst.2007; 99:1435-40) with antigens that can be targeted in certain Cancer types, such as prostate Cancer (Maitland et al, Ernst Schering Foundation. Sympos. Proc.2006; 5:155-79), CD133 in non-small cell lung Cancer (Donnenberg et al, J.Control Release 2007; 122(3):385-91) and glioblastoma (Beier et al, Cancer Res.2007; 67(9):4010-5), and colorectal Cancer (Dalerba et al, Proc.Natl. Acad.Sci.USA 2007; 104) (24)10158-63), pancreatic Cancer (Li et al, Cancer Res.2007; 1030-7) and Prime. Sci.2007; Prime. USA.26; Proc. 44; USA 3-26; Proc. Natl.2007; Proc.9, USA).

For multiple myeloma treatment, suitable targeting antibodies have been described for example against CD38 and CD138(Stevenson, Mol Med 2006; 12(11-12): 345-; Tassone et al, Blood 2004; 104(12):3688-96), CD74(Stein et al, ibid.), CS1(Tai et al, Blood 2008; 112(4):1329-37) and CD40(Tai et al, 2005; Cancer Res.65(13): 5898-; 5906).

Macrophage Migration Inhibitory Factor (MIF) is an important regulator of innate and adaptive immunity and apoptosis. CD74 has been reported to be an endogenous receptor for MIF (Leng et al, 2003, J Exp Med 197: 1467-76). The therapeutic effect of antagonist anti-CD 74 antibodies on MIF-mediated intracellular pathways is useful in the treatment of a wide range of disease states such as bladder Cancer, prostate Cancer, breast Cancer, lung Cancer, colon Cancer and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al, 2004, BMC Cancer 12: 34; Shachar & Haran,2011, Leuk Lymphoma 52: 1446-54); autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (Morand & Leech,2005, Front Biosci 10: 12-22; Shachar & Haran,2011, Leuk Lymphoma 52: 1446-54); kidney diseases such as renal allograft rejection (Lan,2008, Nephron Exp nephrol.109: e 79-83); and many inflammatory diseases (Meyer-Siegler et al, 2009, Mediators inflamepub mar.22, 2009; Takahashi et al, 2009, RespirRes 10: 33; Milatuzumab (hLLl) is an exemplary anti-CD 74 antibody for therapeutic use in treating MIF-mediated diseases.

anti-TNF-alpha antibodies are known in the art and may be used to treat immune disorders, such as autoimmune diseases, immune dysfunction (e.g., graft versus host disease, organ transplant rejection) or diabetes. Known antibodies to TNF- α include the human antibody CDP571(Ofei et al, 2011, Diabetes 45: 881-85); murine antibodies MTNFAI, M2TNFAI, M3 tnfaibi, M302B, and M303(Thermo Scientific, Rockford, il.); infliximab (Centocor, Malvern, Pa.); certolizumab ozogamicin (UCB, Brussels, Belgium); and adalimumab (Abbott, Abbott Park, il.). These and many other known anti-TNF-alpha antibodies can be used in the claimed methods and compositions. Other antibodies useful for treating immune disorders or autoimmune diseases include, but are not limited to, anti-B cell antibodies, such as, for example, trastuzumab, epratuzumab, milnacumab, or hL 243; toslizumab (anti-IL-6 receptor); basiliximab (anti-CD 25); darlizumab (anti-CD 25); efletuzumab (anti-CD 11 a); Moluumab-CD 3 (anti-CD 3 receptor); anti-CD 40L (UCB, Brussels, Belgium); natalizumab (anti- α 4 integrin) and omalizumab (anti-IgE).

The study of checkpoint inhibitor antibodies for cancer therapy has resulted in unprecedented response rates in cancers previously thought to be resistant to cancer therapy (see, e.g., Ott & Bhardwaj,2013, Frontiers in Immunology4: 346; Menzies & Long,2013, the R Adv Med Oncol 5: 278-85; Pardol, 2012, Nature Reviews 12: 252-. Treatment with antagonistic checkpoint blocking antibodies against CTLA-4, PD-1 and PD-L1 is one of the most promising new approaches to immunotherapy of cancer and other diseases. In contrast to most anti-cancer drugs, checkpoint inhibitors do not target tumor cells directly, but rather target lymphocyte receptors or their ligands to enhance the endogenous anti-tumor activity of the immune system (pardol, 2012, Nature Reviews 12: 252-. Because these antibodies act primarily by modulating the immune response to disease cells, they can be used in combination with other therapeutic modalities, such as the anti-HLA-DR antibodies of the invention, to enhance their anti-tumor effects.

Programmed cell death protein 1(PD-1, also known as CD279) encodes a cell surface membrane protein of the immunoglobulin superfamily expressed in B cells and NK cells (Shinohara et al, 1995, Genomics 23: 704-6; Blank et al, 2007, Cancer Immunol Immunother56: 739-45; Finger et al, 1997, Gene 197: 177-87; Pardol, 2012, Nature Reviews 12: 252-. anti-PD 1 antibodies have been used to treat melanoma, non-small cell lung Cancer, bladder Cancer, prostate Cancer, colorectal Cancer, head and neck Cancer, triple negative breast Cancer, leukemia, lymphoma, and renal cell carcinoma (Topalian et al, 2012, N Engl J Med 366: 2443-54; Lipson et al, 2013, Clin Cancer Res19: 462-8; Berger et al, 2008, Clin Cancer Res 14: 3044-51; Gildener-Leapman et al, 2013, Oral Oncol 49: 1089-96; Menzies & Long,2013, Ther Adv Med Oncol 5: 278-85).

Exemplary anti-PD 1 antibodies include pembrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), and ipilimumab (CT-011, CURETECH LTD.). anti-PD 1 antibodies are commercially available, e.g., from

(AB137132)、

(EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH 4).

Programmed cell death 1 ligand 1(PD-L1, also known as CD274) is a ligand for PD-1 and is present on activated T cells, B cells, bone marrow cells, and macrophages. Complexes of PD-1 and PD-L1 inhibit proliferation of CD8+ T cells and reduce immune responses (Topalian et al, 2012, N Engl J Med 366: 2443-54; Brahmer et al, 2012, N Eng J Med 366: 2455-65). anti-PDL 1 antibodies have been used to treat non-small cell lung Cancer, melanoma, colorectal Cancer, renal cell carcinoma, pancreatic Cancer, gastric Cancer, ovarian Cancer, breast Cancer, and hematological malignancies (Brahmer et al, N Eng J Med 366: 2455-65; Ott et al, 2013, Clin Cancer Res19: 5300-9; Radvanyi et al, 2013, Clin Cancer Res19: 5541; Menzies & Long,2013, Ther Adv Med Oncol 5: 278-85; Berger et al, 2008, Clin Cancer Res 14: 13044-51).

Exemplary anti-PDL 1 antibodies include MDX-1105(MEDAREX), MEDI4736(MEDIMMUNE) MPDL3280A (GENENTECH), and BMS-936559(BRISTOL-MYERS SQUIBB). anti-PDL 1 antibodies are also commercially available, for example from AFFYMETRIX EBIOSCIENCE (MIH 1).

Cytotoxic T lymphocyte antigen 4(CTLA-4, also known as CD152) is also a member of the immunoglobulin superfamily that is expressed only on T cells. CTLA-4 is used to inhibit T cell activation and has been reported to inhibit helper T cell activity and enhance regulatory T cell immunosuppressive activity (pardol, 2012, Nature Reviews 12: 252-. anti-CTL 4A antibodies have been used in clinical trials for the treatment of melanoma, prostate Cancer, small cell lung Cancer, non-small cell lung Cancer (Robert & Ghiringhelli,2009, Oncoloist 14: 848-61; Ott et al, 2013, Clin Cancer Res19: 5300; Weber,2007, Oncoloist 12: 864-72; Wada et al, 2013, J Transl Med 11: 89).

Exemplary anti-CTLA-4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). anti-PD 1 antibodies are commercially available, e.g., from

(AB134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H) and THERMO SCIENTIFIC PIERCE (PA5-29572, PA5-23967, PA5-26465, MA1-12205, MA 1-35914). Ipilimumab recently received FDA approval for the treatment of metastatic melanoma (Wada et al, 2013, J trans Med 11: 89).

These and other known checkpoint inhibitor antibodies can be used alone in combination with anti-HLA-DR antibodies or further in combination with interferons, such as interferon- α, for improved cancer therapy.

One of ordinary skill will recognize that any number of antibodies can be raised against a known and well characterized target antigen (e.g., human HLA-DR). Human HLA-DR antigens have been well characterized in the art, for example by their amino acid sequence (see, e.g., GenBank accession number ADM 15723.1).

Bispecific and multispecific antibodies

Bispecific antibodies are useful in many biomedical applications. For example, a bispecific antibody with a binding site for a tumor cell surface antigen and for a T cell surface receptor can direct T cell lysis of a particular tumor cell. Bispecific antibodies that recognize CD3 epitopes on gliomas and T cells have been successfully used to treat brain tumors in human patients (Nitta et al Lancet.1990; 355: 368-371). A preferred bispecific antibody is an anti-CD 3X anti-CD 19 antibody. In alternative embodiments, the anti-CD 3 antibody or fragment thereof may be linked to an antibody or fragment against another B cell-associated antigen, such as anti-CD 3X anti-HLA-DR. In certain embodiments, the techniques and compositions disclosed herein for conjugation of therapeutic agents can be used as targeting moieties with bispecific or multispecific antibodies.

Many methods of producing bispecific or multispecific antibodies are known, for example, as disclosed in U.S. Pat. No. 7,405,320, the examples of which are incorporated herein in part by reference. Bispecific antibodies can be produced by the quadrivalent tumor (quadroma) method, which involves the fusion of two different hybridomas each producing a monoclonal antibody that recognizes a different antigenic site (Milstein and Cuello, Nature, 1983; 305: 537) -540).

Another method for generating bispecific antibodies uses a heterobifunctional cross-linker to chemically link two different monoclonal antibodies (Staerz et al Nature, 1985; 314: 628-. Bispecific antibodies can also be produced by: each of the two parental monoclonal antibodies was reduced to the corresponding half molecule and then mixed and re-oxidized to obtain hybrid structures (Staerz and Bevan. Proc Natl Acad Sci USA.1986; 83: 1453-. Another alternative involves chemically cross-linking two or three separately purified Fab' fragments using a suitable linker. (see, for example, European patent application 0453082).

Other methods include increasing the efficiency of hybridoma production by: transfer of different selectable marker genes into each parental hybridoma by a retrovirus-derived shuttle vector, which subsequently fuses (DeMonte et al Proc Natl Acad Sci USA 1990,87: 2941-; or by transfecting hybridoma cell lines with expression plasmids containing the heavy and light chain genes for different antibodies.

Homologous V can be joined by a peptide linker (usually consisting of more than 12 amino acid residues) of suitable composition and length _H And V _L (iii) a domain to form a single chain fv (scFv) having binding activity. Methods of making scfvs are disclosed in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,132,405, examples portions of each of which are incorporated herein by reference. Reducing the peptide linker length to less than 12 amino acid residues prevents V on the same chain _H And V _L Pairing of domains and forcing V _H And V _L The domains pair with complementary domains on other strands, resulting in the formation of functional multimers. V linked by a linker of 3 to 12 amino acid residues _H And V _L The polypeptide chains of the domains form predominantly dimers (called diabodies). With linkers of 0 to 2 amino acid residues, trimers (called trisomers) and tetramers (called tetrabodies) are advantageous, but apart from linker length, the exact mode of oligomerization appears to depend on the composition and orientation of the V-domain (V-domain) _H -linker-V _L Or V _L -linker-V _H )。

These techniques for generating multispecific or bispecific antibodies present various difficulties in terms of low yields, the need for purification, low stability, or labor intensive techniques. Recently, the so-called DOCK-AND-

To produce virtually any desired combination of antibodies, antibody fragments, and other effector molecules (see, e.g., U.S. patent nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070; 7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398; 8,003,111; and 8,034,352, the respective examples of which are incorporated herein by reference in their entirety). TheThe technology utilizes complementary protein binding domains, called Anchoring Domains (AD) and Dimerization and Docking Domains (DDD), that bind to each other and allow the assembly of complex structures, including dimers, trimers, tetramers, pentamers and hexamers. They form stable complexes in high yield without extensive purification. The DNL technique allows the assembly of monospecific, bispecific or multispecific antibodies. Any technique known in the art for making bispecific or multispecific antibodies may be used in the practice of the claimed methods.

In various embodiments, a conjugate as disclosed herein can be part of a complexed multispecific antibody. Such antibodies may contain two or more different antigen binding sites, with different specificities. Multispecific complexes may bind different epitopes of the same antigen, or may bind two different antigens.

Such antibodies need not only be used in combination, but can be combined into various forms of fusion proteins, such as IgG, Fab, scFv, etc., as described in: U.S. patent nos. 6,083,477; 6,183,744 and 6,962,702 and U.S. patent application publication nos

20030124058；20030219433；20040001825；20040202666；20040219156；20040219203；

20040235065；20050002945；20050014207；20050025709；20050079184；20050169926；

20050175582, respectively; 20050249738, respectively; 20060014245 and 20060034759 of the group consisting of,

the examples of each are incorporated herein in part by reference.

DOCK AND

In certain embodiments, anti-HLA-DR antibodies or fragments can be incorporated into multimeric complexes, for example using the antibody known as DOCK-AND-

The technique of (1). The method utilizes the modulation of cAMP-dependent Protein Kinase (PKA)Specific protein/protein interactions occur between the (R) subunit and the Anchored Domain (AD) of the A Kinase Anchored Protein (AKAP) (Bailie et al, FEBS letters.2005; 579: 3264.Wong and Scott, nat. Rev. mol. cell biol.2004; 5: 959). PKA was first isolated in 1968 from skeletal muscle (Walsh et al, J.biol.chem.1968; 243: 3763), which plays a central role in one of the most well studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunit. The structure of the holoenzyme consists of two catalytic subunits that are maintained in an inactive form by the R subunit (Taylor, J.biol.chem.1989; 264: 8443). The isozymes of PKA were found to have two types of R subunits (RI and RII), and each type has alpha and beta isoforms (Scott, Pharmacol. Ther.1991; 50: 123). The R subunit is only isolated as a stable dimer, and the dimerization domain has been shown to consist of the first 44 amino terminal residues (Newlon et al, nat. struct. biol. 1999; 6: 222). Binding of cAMP to the R subunit results in the release of the active catalytic subunit for a broad spectrum of serine/threonine kinase activity, which is targeted to the selected substrate by compartmentalization of PKA by docking with AKAP (Scott et al, J.biol.chem.1990; 265; 21561).

Since the first AKAP microtubule-associated protein-2 was characterized in 1984 (Lohmann et al, proc.natl.acad.sci. usa.1984; 81:6723), over 50 AKAPs with different structures have been identified in yeast to mammalian species, which are localized to various subcellular sites including the plasma membrane, actin cytoskeleton, nucleus, mitochondria and endoplasmic reticulum (Wong and Scott, nat.rev.mol.cell biol.2004; 5: 959). The AD of AKAP used in PKA is a 14-18 residue amphipathic helix (Carr et al, J.biol.chem.1991; 266:14188) which is reported to have a binding affinity of 2 to 90nM to RII dimer (Alto et al, Proc.Natl.Acad.Sci.USA.2003; 100: 4445). Interestingly, AKAP binds only to the dimeric R subunit. For human RII α, AD binds to a hydrophobic surface formed by 23 amino-terminal residues (Collidge and Scott, Trends Cell biol.1999; 6: 216). Thus, both the dimerization domain and the AKAP binding domain of human RII α are located within the same N-terminal 44 amino acid sequence (Newlon et al, nat. struct. biol. 1999; 6: 222; Newlon et al, EMBO J. 2001; 20:1651), which is herein referred to as DDD.

Human RII alpha DDD and AKAP AD as linker modules

We have developed a platform technology to utilize the DDD of human RII α and the AD of AKAP proteins as a pair of superior linker modules for docking any two entities (hereinafter referred to as a and B) into a non-covalent complex that can be further locked into a stable tethered structure by introducing cysteine residues in DDD and AD at strategic positions to promote disulfide bond formation. The general method of the "dock-and-lock" method is as follows. Entity a is constructed by linking the DDD sequence to a precursor of a, resulting in a first component, which is referred to hereinafter as a. Since the DDD sequence will effect the spontaneous formation of dimers, A will be derived from a ₂ And (4) forming. Entity B is constructed by linking the AD sequence to a precursor of B, thereby generating a second component, which is referred to as B below. a is ₂ The dimeric motif of DDD contained in (a) will create a docking site for binding to the AD sequence contained in (b), thereby facilitating a ₂ And b are prepared to associate to form a group consisting of ₂ b is a binary trimer complex. This binding event is made irreversible by a subsequent reaction to covalently immobilize the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration, since the initial binding interaction should bring the reactive thiol groups on DDD and AD close (Chimura et al, proc.natl.acad.sci.usa.2001; 98:8480) to site-specifically link.

In some preferred embodiments, the anti-HLA-DRMAb DNL constructs may be based on a ₂ b structural variations in which an IgG immunoglobulin molecule (e.g., hL243) is linked at its C-terminus to two copies of the AD moiety. Preferably, an AD moiety is attached to the C-terminus of each light chain. Each AD moiety is capable of binding to two DDD moieties in the form of a dimer. By attaching a cytokine or other therapeutic protein or peptide to each DDD moiety, four copies of the cytokine or other therapeutic moiety will be conjugated to each IgG molecule.

This site-specific linkage is also expected to retain the original activity of the two precursors by attaching DDD and AD away from the functional groups of the two precursors. This approach is modular in nature and potentially applicable to site-specific and covalent attachment of a wide range of substances. The DNL process is disclosed in U.S. patent nos. 7,550,143; 7,521,056, respectively; 76,534,866, respectively; 7,527,787 and 7,666,400, the respective examples of which are incorporated herein by reference in their entirety.

In some preferred embodiments, the effector moiety is a protein or peptide, more preferably an antibody, antibody fragment or cytokine, which may be linked to a DDD or AD unit to form a fusion protein or peptide. Various methods of preparing fusion proteins are known, including nucleic acid synthesis, hybridization and/or amplification, to produce synthetic double-stranded nucleic acids encoding the fusion proteins of interest. Such double-stranded nucleic acids can be inserted into expression vectors by standard Molecular biology techniques to produce fusion proteins (see, e.g., Sambrook et al, Molecular Cloning, A laboratory manual, 2 nd edition, 1989). In these preferred embodiments, the AD and/or DDD moieties may be linked to the N-terminus or C-terminus of the effector protein or peptide. However, the skilled person will recognise that the attachment site of an AD or DDD moiety on the effector moiety may vary, depending on the chemical nature of the effector moiety and the moiety with which the effector moiety participates in its physiological activity. Site-specific attachment of various effector moieties can be performed using techniques known in the art, such as using divalent cross-linkers and/or other chemical conjugation techniques.

DDD and AD sequence variants

In certain embodiments, the AD and DDD sequences incorporated into the anti-HLA-DR MAb DNL complex comprise the amino acid sequences of DDD1(SEQ ID NO:7) and AD1(SEQ ID NO:9) below. In a more preferred embodiment, the AD and DDD sequences comprise the amino acid sequences of DDD2(SEQ ID NO:8) and AD2(SEQ ID NO:10) designed to promote disulfide bond formation between DDD and AD moieties.

DDD1

DDD2

AD1

AD2

However, in alternative embodiments, sequence variant AD and/or DDD moieties may be used to construct anti-HLA-DR MAb DNL complexes. The structure-function relationship of the AD and DDD domains has been the subject of research. (see, e.g., Burns-Hamuro et al, 2005, Protein Sci 14: 2982-92; Carr et al, 2001, J Biol Chem 276: 17332-38; Alto et al, 2003, Proc Natl Acad Sci USA 100: 4445-50; Hundsrucker et al, 2006, Biochem J396:297-

For example, Kinderman et al (2006) examined the crystal structure of AD-DDD binding interactions and concluded that the human DDD sequence contains many conserved amino acid residues important for dimer formation or AKAP binding, underlined below in SEQ ID NO:7 (see FIG. 1 of Kinderman et al, 2006, incorporated herein by reference). The skilled person will recognize that in designing sequence variants of DDD sequences it is desirable to avoid changes in any underlined residues, whereas conservative amino acid substitutions may be made for residues that are less important for dimerization and AKAP binding. Conservative amino acid substitutions are discussed in more detail below, but may involve, for example, substitution of an aspartic acid residue for a glutamic acid residue, or substitution of a leucine or valine residue for an isoleucine residue, and the like. Such conservative amino acid substitutions are well known in the art.

Human DDD sequences from protein kinase A

Alto et al (2003) performed bioinformatic analyses of the AD sequences of various AKAP proteins, designed a RII-selective AD sequence called AKAP-IS (SEQ ID NO:9) with a binding constant of 0.4nM for DDD. The AKAP-IS sequence was designed as a peptide antagonist of the binding of AKAP to PKA. The residues in the AKAP-IS sequence that tend to reduce binding to DDD are underlined in SEQ ID NO 9.

AKAP-IS sequence

Similarly, Gold (2006) utilized crystallography and peptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:11) that exhibited 5 orders of magnitude higher selectivity for the RII isoform of PKA as compared to the RI isoform. Underlined residues indicate positions relative to amino acid substitutions of the AKAP-IS sequence that improve binding to the DDD portion of RII α. In this sequence, the N-terminal Q residue is numbered residue number 4 and the C-terminal A residue is numbered residue number 20. Residues that can be substituted to affect affinity for RII α are residues 8, 11, 15, 16, 18, 19 and 20(Gold et al, 2006). It IS contemplated that in certain alternative embodiments, the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD partial sequence to prepare anti-HLA-DRMAb DNL constructs. It IS contemplated that the AD moiety may also comprise additional N-terminal residues of cysteine and glycine and C-terminal residues of glycine and cysteine, as shown in SEQ ID NO:10, as well as the AKAP-IS sequence shown in SEQ ID NO: 9.

SuperAKAP-IS

Hundsrucker et al (2006) also developed additional peptide competitors of AKAP binding to PKA with binding constants as low as 0.4nM to DDD, the RII form of PKA. The sequences of various AKAP antagonist peptides are provided in Table 1 of Hundsrucker et al (incorporated herein by reference). Highly conserved residues in the AD domains of different AKAP proteins are indicated below by underlining with reference to the AKAP IS sequence (SEQ ID NO: 9). The residues were identical to those observed by Alto et al (2003) except that a C-terminal alanine residue was added. (see FIG. 4 of Hundsrucker et al (2006), which is incorporated herein by reference.)

AKAP-IS

Carr et al (2001) examined the degree of sequence homology between different AKAP-binding DDD sequences from human and non-human proteins and identified the residues in the DDD sequence that appear to be most highly conserved between different DDD moieties. These are indicated below by underlining with reference to the human PKA RII α DDD sequence of SEQ ID NO 7. Particularly conserved residues are further indicated in italics. Residues overlap, but are not identical, to those indicated by Kinderman et al (2006) to be important for binding to AKAP proteins.

Those skilled in the art will recognize that, in general, those amino acid residues that are highly conserved among DDD and AD sequences from different proteins are those that preferably remain constant when amino acid substitutions are made, while less highly conserved residues may be more readily altered to produce sequence variants of the AD and/or DDD sequences described herein.

Allotype of antibody

The immunogenicity of therapeutic antibodies is associated with an increased risk of infusion reactions and a decreased duration of therapeutic reactions (Baert et al, 2003, N Engl J Med 348: 602-08). The extent to which a therapeutic antibody induces an immune response in a host may be determined in part by the allotype of the antibody (Stickler et al, 2011, Genes and Immunity 12: 213-21). Antibody allotypes are associated with amino acid sequence variations at specific positions in the antibody constant region sequence. The isotype of IgG antibodies containing the heavy chain gamma-type constant region was designated the Gm isotype (1976, J Immunol 117: 1056-59).

For the common IgG1 human antibody, the most common allotype is G1m1(Stickler et al, 2011, Genes and Immunity 12: 213-21). However, the G1m3 allotype also frequently occurs in caucasians (supra). The G1m1 antibody is reported to contain allotypic sequences (supra) that tend to induce an immune response when administered to a non-G1 m1(nG1ml) recipient (e.g., a G1m3 patient). non-G1 m1 allotypic antibodies were not immunogenic when administered to G1m1 patients (supra).

The human G1m1 allotype comprises amino acids D12(Kabat position 356) and L14(Kabat position 358) in the CH3 sequence of heavy chain IgG 1. The nG1M1 allotype contains the amino acids E12 and M14 at the same position. Both the G1m1 and nG1m1 allotypes contain an E13 residue between the two variable sites, and the allotypes are sometimes referred to as DEL and EEM allotypes. Non-limiting examples of heavy chain constant region sequences for the exemplary antibodies rituximab (SEQ ID NO:12) and veltuzumab (SEQ ID NO:13) are shown for the G1m1 and nG1m1 allotypes of antibodies.

Rituxib single-antibody heavy chain variable region sequence (SEQ ID NO:12)

Vituzumab heavy chain variable region (SEQ ID NO:13)

With respect to therapeutic antibodies, veltuzumab and rituximab are humanized and chimeric IgG1 antibodies, respectively, against CD20 for the treatment of various hematologic malignancies and/or autoimmune diseases. Table 1 compares the allotypic sequences of rituximab and veltuzumab. As shown in table 1, rituximab (G1m17,1) is a DEL allotype IgG1 with additional sequence variation at Kabat position 214 (heavy chain CH1), lysine at rituximab, versus arginine in veltuzumab. It has been reported that veltuzumab is less immunogenic in subjects than rituximab (see, e.g., Morchhauser et al, 2009, J Clin Oncol 27: 3346-53; golden enberg et al, 2009, Blood 113: 1062-70; Robak & Robak,2011, BioDrugs 25:13-25), this effect being attributed to the difference between humanized and chimeric antibodies. However, the differences in allotypes between the EEM and DEL allotypes may also be responsible for the lower immunogenicity of the veltuzumab.

TABLE 1 allotypes of rituximab versus veltuzumab

To reduce the immunogenicity of a therapeutic antibody in an individual of nG1ml genotype, it is desirable to select an allotype of the antibody to correspond to the EEM allotype, wherein Kabat position 356 is a glutamic acid residue, Kabat position 358 is a methionine, and Kabat position 214 is preferably an arginine residue. Surprisingly, it was found that repeated subcutaneous administration of the G1m3 antibody for a long time did not result in a significant immune response.

Amino acid substitutions

In alternative embodiments, the disclosed methods and compositions may involve the production and use of proteins or peptides having one or more substituted amino acid residues. For example, for the preparation of

The DDD and/or AD sequences of the constructs may be modified as described above.

One skilled in the art will appreciate that, in general, an amino acid substitution typically involves replacing the amino acid with another amino acid having relatively similar properties (i.e., a conservative amino acid substitution). The nature of the various amino acids and the effect of amino acid substitutions on protein structure and function have been the subject and knowledge of extensive research in the art.

For example, the hydropathic index of amino acids may be considered (Kyte and Doolittle,1982, J.mol.biol.,157: 105-. The relative hydrophilic character of amino acids contributes to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules. Each amino acid is assigned a hydropathic index according to its hydrophobic and charge characteristics (Kyte and Doolittle,1982), which are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In conservative substitution, it is preferred to use amino acids whose hydropathic index is within. + -. 2, more preferably within. + -.1, even more preferably within. + -. 0.5.

Amino acid substitutions can also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554,101). Amino acid residues have been assigned hydrophilicity values: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+ 3.0); glutamic acid (+ 3.0); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5.+ -. 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is preferred to replace the amino acid with another amino acid having a similar hydrophilicity.

Other considerations include the size of the amino acid side chain. For example, it is generally not preferred to replace an amino acid with a compact side chain (e.g., glycine or serine) with an amino acid with a bulky side chain (e.g., tryptophan or tyrosine). The influence of various amino acid residues on the secondary structure of a protein is also a consideration. The influence of different amino acid residues on the tendency of protein domains to adopt alpha-helices, beta-sheets or reverse secondary structures has been determined by empirical studies and is known in the art (see, e.g., Chou & Fasman,1974, Biochemistry,13: 222-cake 245; 1978, Ann. Rev. Biochemistry, 47: 251-cake 276; 1979, Biophys. J.,26: 367-cake 384).

Based on these considerations and extensive empirical studies, conservative amino acid substitution tables have been constructed and are known in the art. For example: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Or: ala (A) leu, ile, val; arg (R) gln, asn, lys; asn (N) his, asp, lys, arg, gln; asp (D) asn, glu; cys (C) ala, ser; gln (Q) glu, asn; glu (E) gln, asp; gly (G) ala; his (H) asn, gln, lys, arg; ile (I) val, met, ala, phe, leu; leu (L) val, met, ala, phe, ile; lys (K) gln, asn, arg; met (M) phe, ile, leu; phe (F) leu, val, ile, ala, tyr; pro (P) ala; ser (S), thr; thr (T) ser; trp (W) phe, tyr; tyr (Y) trp, phe, thr, ser; val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether the residue is located within the protein or exposed to a solvent. For internal residues, conservative substitutions include: asp and Asn; ser and Thr; ser and Ala; thr and Ala; ala and Gly; ile and Val; val and Leu; leu and Ile; leu and Met; phe and Tyr; tyr and Trp (see, e.g., PROWL website rockfiller. For solvent exposed residues, conservative substitutions include: asp and Asn; asp and Glu; glu and Gln; glu and Ala; gly and Asn; ala and Pro; ala and Gly; ala and Ser; ala and Lys; ser and Thr; lys and Arg; val and Leu; leu and Ile; ile and Val; phe and Tyr. Various matrices have been constructed (supra) to aid in the selection of amino acid substitutions, such as the PAM250 scoring matrix, the Dayhoff matrix, the Grantham matrix, the McLachlan matrix, the Doolittle matrix, the Henikoff matrix, the Miyata matrix, the Fitch matrix, the Jones matrix, the Rao matrix, the Levin matrix, and the Risler matrix (supra).

The presence of intermolecular or intramolecular bonds may also be considered in determining amino acid substitutions, such as ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu), or disulfide bonds between adjacent cysteine residues.

Methods for substituting any amino acid for any other amino acid in the encoded protein sequence are well known and are a matter of routine experimentation by those skilled in the art, for example by site-directed mutagenesis techniques or by synthesizing and assembling oligonucleotides encoding amino acid substitutions and splicing into expression vector constructs.

Conjugation schemes

In certain embodiments, the anti-HLA-DR antibody or fragment may be conjugated to one or more therapeutic or diagnostic agents. The therapeutic agents need not be the same, but may be different, for example, drugs and radioisotopes. For example, ¹³¹ i can be incorporated into tyrosine as well as drugs linked to the epsilon amino group of lysine residues of antibodies or fusion proteins. Therapeutic and diagnostic agents may also be attached, for example, to the reduced SH groups and/or to the carbohydrate side chains. Many methods for preparing covalent or non-covalent conjugates of therapeutic or diagnostic agents and antibodies or fusion proteins are known in the art, and any such known method may be used.

The therapeutic or diagnostic agent may be linked to the hinge region of the reduced antibody component by disulfide bond formation. Alternatively, heterobifunctional cross-linkers such as N-succinyl 3- (2-pyridyldithio) propionate (SPDP) may be used to link these reagents. Yu et al, int.J.cancer 56:244 (1994). General techniques for such conjugation are well known in the art. See, e.g., Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); updalacis et al, "Modification of Antibodies by Chemical Methods," MONOCLONAL ANTIBODIES: PRINCIPLES AND application, Birch et al (ed.), page 187-230 (Wiley-Liss, Inc.1995); price, "Production AND Characterization of Synthetic Peptide-Derived Antibodies," MONOCLONAL Antibodies: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al (eds.), pages 60-84 (Cambridge University Press 1995). Alternatively, the therapeutic or diagnostic agent may be conjugated through a carbohydrate moiety in the Fc region of the antibody. Carbohydrate groups can be used to increase the loading of the same agent bonded to the thiol, or carbohydrate moieties can be used to bond different therapeutic or diagnostic agents.

Methods for conjugating peptides to antibody components via the carbohydrate moiety of an antibody are well known to those skilled in the art. See, e.g., Shih et al, int.J. cancer 41:832 (1988); shih et al, int.J.cancer46: 1101 (1990); and Shih et al, U.S. Pat. No. 5,057,313, which is incorporated herein by reference in its entirety. The general method involves reacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine functional group. This reaction produces an initial schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

If the antibody used as the antibody component of the immunoconjugate is an antibody fragment, the Fc region may not be present. However, carbohydrate moieties may be introduced into the light chain variable region of a full-length antibody or antibody fragment. See, e.g., Leung et al, J.Immunol.154:5919 (1995); hansen et al, U.S. Pat. No. 5,443,953(1995), Leung et al, U.S. Pat. No. 6,254,868, which are incorporated herein by reference in their entirety. The engineered carbohydrate moiety is used to attach a therapeutic or diagnostic agent.

In some embodiments, a chelator can be linked to an antibody, antibody fragment, or fusion protein and used to chelate a therapeutic or diagnostic agent, such as a radionuclide. Exemplary chelating agents include, but are not limited to, DTPA (e.g., Mx-DTPA), DOTA, TETA, NETA, or NOTA. Methods of conjugation and attachment of metals or other ligands to proteins using chelators are well known in the art (see, e.g., U.S. patent application serial No. 12/112,289, which is incorporated herein by reference in its entirety).

In certain embodiments, the radioactive metal or paramagnetic ion may be attached to the protein or peptide by reaction with a reagent having a long tail to which multiple chelating groups may be attached for binding the ion. Such tails may be polymers such as polylysine, polysaccharides, or derivatized chains with pendant groups that can be bonded to chelating groups such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, thiosemicarbazones, polyoxins, and similar groups known to be useful for this purpose.

The chelate may be linked directly to an antibody or peptide, such as disclosed in U.S. Pat. No. 4,824,659, which is incorporated by reference in its entiretyIncorporated herein. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, with diagnostic isotopes in the general energy range of 60 to 4,000keV, such as ¹²⁵ I、 ¹³¹ I、 ¹²³ I、 ¹²⁴ I、 ⁶² Cu、 ⁶⁴ Cu、 ¹⁸ F、 ¹¹¹ In、 ⁶⁷ Ga、 ⁶⁸ Ga、 ^99m Tc、 ^94m Tc、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ⁷⁶ Br was used together for radiographic imaging. When complexed with non-radioactive metals (e.g. manganese, iron and gadolinium), the same chelates may be used for MRI. Macrocyclic chelates such as NOTA, DOTA and TETA can be used with radionuclides of various metals and radiometals, most particularly gallium, yttrium and copper. This metal-chelate complex can be made very stable by adjusting the ring size to the target metal. Including other cyclic chelates, e.g. macrocyclic polyethers, which stabilize bound nuclides such as for RAIT ²²³ Ra is significant.

More recently, the use in PET scanning techniques has been disclosed ¹⁸ F-labelling, for example by reacting F-18 with a metal or other atom such as aluminium. ¹⁸ The F-Al conjugate can be complexed with a chelating group such as DOTA, NOTA or NETA, either directly linked to the antibody or used to label the targetable construct in a pretargeting approach. This F-18 tagging technique is disclosed in U.S. patent application serial No. 12/112,289, filed on 30/4/2008, which is incorporated herein by reference in its entirety.

In some preferred embodiments, the conjugation scheme is based on facile thiol-maleimide, thiol-vinylsulfone, thiol-bromoacetamide, or thiol-iodoacetamide reactions at neutral or acidic pH. This eliminates the need for higher pH conditions for conjugation, which is necessary, for example, when using active esters. Further details of exemplary conjugation schemes are described in the examples section below.

Therapeutic treatment

In another aspect, the invention relates to a method of treating a subject comprising administering to the subject a therapeutically effective amount of a therapeutic conjugate as described herein. Diseases that can be treated with the therapeutic conjugates described herein include, but are not limited to, B cell malignancies (e.g., non-hodgkin's lymphoma, mantle cell lymphoma, multiple myeloma, hodgkin's lymphoma, diffuse large B cell lymphoma, burkitt's lymphoma, follicular lymphoma, acute lymphatic leukemia, chronic lymphatic leukemia, hairy cell leukemia) using anti-HLA-DR immunoconjugates. More preferably, the cancer is AML (acute myeloid leukemia), ALL (acute lymphocytic leukemia) or MM (multiple myeloma). However, any HLA-DR positive tumor can be treated with the immunoconjugates of the invention, e.g., skin cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, endometrial cancer, cervical cancer, testicular cancer, melanoma, renal cancer, or liver cancer. These therapeutic agents may be administered once or repeatedly, depending on the disease state and tolerance of the conjugate, and may also be optionally used in combination with other therapeutic modalities, such as surgery, external radiation, radioimmunotherapy, immunotherapy, chemotherapy, antisense therapy, interfering RNA therapy, gene therapy, and the like. Each combination will accommodate the tumor type, stage, patient condition and prior treatment, as well as other factors considered by the administering physician.

As used herein, the term "subject" refers to any animal (i.e., vertebrates and invertebrates), including but not limited to mammals, including humans. The term is not intended to be limited to a particular age or gender. Thus, the term includes adult and newborn subjects as well as fetuses, whether male or female. The dosages given herein are for humans, but may be adjusted to the size of other mammals, as well as children, depending on body weight or square meter size.

In one exemplary embodiment, the hL243 antibody is a humanized antibody comprising the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:1), CDR2(WINTYTREPTYADDFKG, SEQ ID NO:2) and CDR3(DITAVVPTGFDY, SEQ ID NO:3) and the light chain CDR sequences CDR1(RASENIYSNLA, SEQ ID NO:4), CDR2(AASNLAD, SEQ ID NO:5) and CDR3(QHFWTTPWA, SEQ ID NO: 6).

In a preferred embodiment, the antibody for use in the treatment of human disease is a human or humanized (CDR-grafted) form of the antibody; although murine and chimeric forms of the antibody may be used. The same species of IgG molecule is most preferred as a delivery agent to minimize immune responses. This is particularly important when considering repeated treatments. It is unlikely for human, human or humanized IgG antibodies to generate an anti-IgG immune response in a patient. Antibodies such as hLL1 and hLL2 internalize rapidly upon binding to an internalizing antigen on a target cell, meaning that the carried chemotherapeutic drug is also internalized rapidly into the cell. However, antibodies with slower internalization rates can also be used to achieve selective therapy.

In another preferred embodiment, the therapeutic conjugates are useful for treating autoimmune diseases or immune system dysfunction (e.g., graft versus host disease, organ transplant rejection). Antibodies for use in treating autoimmune diseases/immune dysfunction diseases can bind to exemplary antigens, including but not limited to BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD 3679 a, CD79a, CD117, CD138, FMC-7, and HLA-DR. As described above, antibodies that bind to these and other target antigens can be used to treat autoimmune diseases or immune dysfunction diseases. Autoimmune diseases that can be treated with the immunoconjugate can include acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, adenoidal syndrome, bullous pemphigoid, diabetes, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, takayasu arteritis, ANCA-associated vasculitis, addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, sjogren syndrome, primary biliary cirrhosis, hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, multiple sclerosis, and multiple sclerosis, Polychondritis, bullous pemphigoid, pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis or fibrotic alveolitis.

In another preferred embodiment, the therapeutic agent used in combination with the camptothecin conjugates of the invention can comprise one or more isotopes. Radioisotopes useful for treating diseased tissue include, but are not limited to ¹¹¹ In， ¹⁷⁷ Lu， ²¹² Bi， ²¹³ Bi， ²¹¹ At， ⁶² Cu， ⁶⁷ Cu， ⁹⁰ Y， ¹²⁵ I， ¹³¹ I， ³² P， ³³ P， ⁴⁷ Sc， ¹¹¹ Ag， ⁶⁷ Ga， ¹⁴² Pr， ¹⁵³ Sm， ¹⁶¹ Tb， ¹⁶⁶ Dy， ¹⁶⁶ Ho， ¹⁸⁶ Re， ¹⁸⁸ Re， ¹⁸⁹ Re， ²¹² Pb， ²²³ Ra， ²²⁵ Ac， ⁵⁹ Fe， ⁷⁵ Se， ⁷⁷ As， ⁸⁹ Sr， ⁹⁹ Mo， ¹⁰⁵ Rh， ¹⁰⁹ Pd， ¹⁴³ Pr， ¹⁴⁹ Pm， ¹⁶⁹ Er， ¹⁹⁴ Ir， ¹⁹⁸ Au， ¹⁹⁹ Au， ²²⁷ Th and ²¹¹ Pb。

the decay energy of the therapeutic radionuclide is preferably 20 to 6,000keV, preferably 60 to 200keV for Auger emitters, 2,500keV for beta emitters and preferably 4,000 to 6,000keV for alpha emitters. The maximum decay energy of useful beta-emitting species is preferably 20-5,000keV, more preferably 100-4,000keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that decay substantially as Auger emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. The decay energy of useful beta-emitting species is preferably < 1,000keV, more preferably < 100keV, and most preferably < 70 keV. It is also preferred that the radionuclide substantially decays to produce alpha-particle decay.Such radionuclides include, but are not limited to: dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227, and Fm-255. The decay energy of useful alpha-emitting radionuclides is preferably 2,000-10,000keV, more preferably 3,000-8,000keV, and most preferably 4,000-7,000 keV. Other potential radioisotopes which may be used include ¹¹ C， ¹³ N， ¹⁵ O， ⁷⁵ Br， ¹⁹⁸ Au， ²²⁴ Ac， ¹²⁶ I， ¹³³ I， ⁷⁷ Br， ^113m In， ⁹⁵ Ru， ⁹⁷ Ru， ¹⁰³ Ru， ¹⁰⁵ Ru， ¹⁰⁷ Hg， ²⁰³ Hg， ^121m Te， ^122m Te， ^125m Te， ¹⁶⁵ Tm， ¹⁶⁷ Tm， ¹⁶⁸ Tm， ¹⁹⁷ Pt， ¹⁰⁹ Pd， ¹⁰⁵ Rh， ¹⁴² Pr， ¹⁴³ Pr， ¹⁶¹ Tb， ¹⁶⁶ Ho， ¹⁹⁹ Au， ⁵⁷ Co， ⁵⁸ Co， ⁵¹ Cr， ⁵⁹ Fe， ⁷⁵ Se， ²⁰¹ Tl， ²²⁵ Ac， ⁷⁶ Bt， ¹⁶⁹ Yb, and the like.

Radionuclides and other metals may be delivered, for example, using a chelating group attached to an antibody or conjugate. Macrocyclic chelates such as NOTA, DOTA and TETA are useful for a variety of metals and radioactive metals, most particularly radionuclides of gallium, yttrium and copper. Such metal-chelate complexes can be made very stable by adjusting the ring size to the target metal. Other cyclic chelates may be used, e.g. for complexation ²²³ Macrocyclic polyethers of Ra.

Therapeutic agents for use in combination with the camptothecin conjugates described herein also include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and pro-apoptotic agents, particularly doxorubicin, methotrexate, paclitaxel, other camptothecins, and other analogs from these and other classes of anticancer agents. Other cancer chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable chemotherapeutic agents are described in REMINGTON 'S PHARMACEUTICAL SCIENCES, 19 th edition (Mack Publishing co.1995), and GOODMAN AND GILMAN' S phe PHARMACOLOGICAL bases OF THERAPEUTICS, 7 th edition (millan Publishing co.1985), as well as revisions OF these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those skilled in the art.

Exemplary drugs for use include, but are not limited to, 5-fluorouracil, afatinib, aplidine (aplidine), azaribine (azaribine), anastrozole (anastrozole), anthracyclines, axitinib (axitinib), AVL-101, AVL-291, bendamustine (bendamustine), bleomycin (bleomycin), bortezomib (bortezomib), bosutinib (bosutinib), bryostatin-1 (bryostatin-1), busulfan, calicheamicin (calicheamicin), camptothecin (camptothecin), carboplatin, 10-hydroxycamptothecin, carmustine (carmustine), celecoxib (celecoxib), chlorambucil (loraucil), cisplatin (CDmbDP), Cox-2 inhibitors, irinotecan (irinotecan), cricotine-11, CPatinib (CPnothiazide), capreotinib (CPsinx), capreotide (CPsinx-38), capreotide (capreotide, capreotide (capreotide), capreotide (e), capreotide (e), capreotide (doxib), doxycycline (doxycycline), doxycycline (doxycycline), doxycycline (doxycycline), capreotide (doxycycline), doxycycline (doxycycline), capreotide (doxycycline (cprinone), doxycycline), doxycycline (cprinone, doxycycline (cprinone (cp, dinaciclib, docetaxel, dactinomycin, doxorubicin, 2-pyrrolindirubicin (2P-DOX), cyanomorpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epipodophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3',5' -O-dioleoyl-FudR (FUdR-dO), fludarabine (fludarabine), flutamide, farnesyl protein transferase inhibitors, fusiridol, etatinib, gatapib, gaC-4, GS-0834, Gemcitabine, Adriant, Gemcitabine, Geranitidine, Gemcitabine, Fovatinib, Gemcitabine, idarubicin, ifosfamide, imatinib, L-asparaginase, lapatinib, lenalidomide (linolidamide), leucovorin, LFM-a13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosourea (nitrosula), olapananib, lincomycin (plicomycin), procarbazine, paclitaxel, PCI-32765, pentostatin (pentostatin), PSI-341, raloxifene, semustine, sorafenib, streptozotocin, SU 48, sunitinib, tamoxifen (tamoxifen), temozolomide (temozolomide), topotecan (topotecan), topotecan, trogoptermide (tioxaparin), and mixtures thereof, Uracil mustard (uracil mustard), vatalanib (vatalanib), vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD 1839. These agents may be part of the conjugates described herein, or may be administered in combination with the conjugates before, simultaneously with, or after the conjugates. Alternatively, one or more therapeutic naked antibodies known in the art may be used in combination with the conjugate. Exemplary therapeutic naked antibodies are described above.

In a preferred embodiment, the therapeutic agent used in combination with the DNA-blocking antibody conjugate (e.g., SN-38-ADC) is a microtubule inhibitor, such as a vinca alkaloid, a taxane, a maytansinoid (maytansinoid), or an auristatin (auristatin). Exemplary known microtubule inhibitors include paclitaxel, vincristine, vinblastine, mertansine, epothilone, docetaxel, discodermolide, combretastatin, podophyllotoxin, CI-980, phenylethanoids, eleutherobin (stepanacins), curcutins, 2-methoxyestradiol, E7010, methoxybenzenesulfonamide, vinorelbine, vinflunine, vindesine, dolastatins (dolastatins), spongistatin, rhizomycin (rhizoxin), tasidotatin, halichondrins (halicodins), hemins (hemiasterins), cryptophycin (cryptophycin)52, MMMAE, and eribulin mesylate (eribulin mesylate).

In another preferred embodiment, the therapeutic agent used in combination with a DNA-blocking ADC (e.g., SN-38-antibody conjugate) is a PARP inhibitor, such as olaparib, talazoparib (BMN-673), rucaparib (rucaparib), veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699, BSI-201, CEP-8983, or 3-aminobenzamide.

In another alternative, the therapeutic agent used in combination with the antibody or immunoconjugate is a bruton kinase inhibitor, such as ibrutinib (PCI-32765), PCI-45292, CC-292(AVL-292), ONO-4059, GDC-0834, LFM-A13, or RN 486.

In another alternative, the therapeutic agent used in combination with the antibody or immunoconjugate is a PI3K inhibitor, such as Idelalisib, Wortmannin (Wortmannin), desmethoxyviridin (demethoxyviridin), perifosine (perifosine), PX-866, IPI-145(duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE477, CUDC-907, ZAEZ-136, or LY 294002.

Therapeutic agents that can be used with camptothecin conjugates can also comprise a toxin conjugated to a targeting moiety. Toxins that may be used in this regard include ricin (ricin), abrin (abrin), ribonuclease (rnase), ranpirnase (ranpirnase), dnase I, staphylococcal enterotoxin-a, pokeweed antiviral protein (pokeweed antiviral protein), gelonin (gelonin), diphtheria toxin, pseudomonas exotoxin and pseudomonas endotoxin. (see, e.g., Pastan et al, Cell (1986),47:641, and Sharkey and Goldenberg, CA cancer JClin.2006 Jul-Aug; 56(4): 226-43.) other toxins suitable for use herein are known to those of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Another class of therapeutic agents may include one or more immunomodulatory agents. The immunomodulator used may be selected from cytokines, stem cell growth factors, lymphotoxins, hematopoietic factors, Colony Stimulating Factors (CSF), Interferons (IFN), erythropoietins, thrombopoietins and combinations thereof. Particularly useful are lymphotoxins, such as Tumor Necrosis Factor (TNF); hematopoietic factors, such as Interleukins (IL); colony stimulating factors, such as granulocyte colony stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF); interferons, such as interferon- α, - β, - γ or- λ; and stem cell growth factors such as those known as "SI factor". Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; (ii) insulin; proinsulin; relaxin; relaxin original; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH); a liver growth factor; prostaglandins, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substances (mullerian-inhibiting substance); mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); an osteoinductive factor; interferons, such as interferon- α, - β, and- γ; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and Lymphotoxin (LT). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

Chemokines used include RANTES, MCAF, MIP 1-alpha, MIP 1-beta and IP-10.

One of ordinary skill in the art will recognize that immunoconjugates of the invention comprising camptothecin conjugated to an antibody or antibody fragment can be used alone or in combination with one or more other therapeutic agents. Such as a second antibody, a second antibody fragment, a second immunoconjugate, a radionuclide, a toxin, a drug, a chemotherapeutic agent, radiation therapy, a chemokine, a cytokine, an immunomodulator, an enzyme, a hormone, an oligonucleotide, RNAi or siRNA. These other therapeutic agents may be administered separately from, in combination with, or attached to the antibody-drug immunoconjugate of the invention.

Formulation and administration

Suitable routes of administration for the conjugates include, but are not limited to, oral, parenteral, subcutaneous, rectal, transmucosal, enteral, intramuscular, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injection. The preferred route of administration is parenteral. Alternatively, the compound may be administered in a local rather than systemic manner, for example, by direct injection of the compound into a solid tumor.

The immunoconjugates can be formulated according to known methods to prepare pharmaceutically useful compositions, wherein the immunoconjugate is combined in admixture with a pharmaceutically suitable excipient. Sterile phosphate buffered saline is one example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art. See, for example, Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5 th edition (Lea & Febiger 1990), AND Gennaro (eds.), REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition (Mack Publishing Company1990) AND revisions thereof.

In a preferred embodiment, the immunoconjugate is formulated in Good biological buffers (pH 6-7) using a buffer selected from the group consisting of: n- (2-acetamido) -2-aminoethanesulfonic Acid (ACES); n- (2-acetamido) iminodiacetic acid (ADA); n, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES); 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES); 2- (N-morpholino) ethanesulfonic acid (MES); 3- (N-morpholino) propanesulfonic acid (MOPS); 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO); and piperazine-N, N' -bis (2-ethanesulfonic acid) [ Pipes ]. More preferably the buffer is MES or MOPS, preferably at a concentration of 20 to 100mM, more preferably about 25 mM. Most preferred is 25mM MES, pH 6.5. The formulation may further comprise 25mM trehalose and 0.01% v/v polysorbate 80 as excipients, with the final buffer concentration being changed to 22.25mM as a result of the addition of the excipient. The preferred method of storage is as a lyophilized formulation of the conjugate, stored at a temperature in the range of-20 ℃ to 2 ℃, most preferably 2 ℃ to 8 ℃.

The immunoconjugate may be formulated for intravenous administration, for example by bolus injection, slow infusion, or continuous infusion. Preferably, the antibodies of the invention are infused over a period of less than about 4 hours, more preferably, over a period of less than about 3 hours. For example, the first 25-50mg may be infused over 30 minutes, preferably even 15 minutes, with the remainder infused over the next 2-3 hours. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control the duration of action of the therapeutic conjugate. Controlled release formulations can be prepared by complexing or adsorbing the immunoconjugate using a polymer. For example, biocompatible polymers include poly (ethylene-co-vinyl acetate) matrices and matrices of stearic acid dimer and polyanhydride copolymer of sebacic acid. Sherwood et al, Bio/Technology 10:1446 (1992). The rate of release of the immunoconjugate from such a matrix depends on the molecular weight of the immunoconjugate, the amount of immunoconjugate within the matrix, and the size of the dispersed particles. Saltzman et al, Biophys.J.55:163 (1989); sherwood et al, supra. Other solid dosage forms are described in the following: ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5 th edition (Lea & Febiger 1990), AND Gennaro (eds.), REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition (Mack publishing Company1990), AND revisions thereof.

In general, the dose of immunoconjugate administered to a person will vary depending on factors such as the age, weight, height, sex, general physical state, and past medical history of the patientAnd (4) transforming. For example, the dose of 1-20mg/kg may be 70-1,400mg for a 70kg patient, or 41-824mg/m for a 1.7m patient ² . The dosage may be repeated as desired, for example, once a week for 4-10 weeks, once a week for 8 weeks, or once a week for 4 weeks. It may also be administered less frequently, for example every other week for months, or monthly or quarterly for months, as needed for maintenance therapy. Preferred doses may include, but are not limited to, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, and 18 mg/kg. The dose is preferably administered multiple times, once or twice weekly. A minimum dose regimen lasting 4 weeks, more preferably 8 weeks, more preferably 16 weeks or more may be used. The administration regimen may comprise once or twice weekly administration in a cycle selected from: (i) weekly; (ii) every other week; (iii) for one week, then stop for two, three or four weeks; (iv) two weeks of treatment, followed by one, two, three or four weeks off; (v) for three weeks, then stop for one, two, three, four or five weeks; (vi) for four weeks of treatment, then discontinued for one, two, three, four or five weeks; (vii) five weeks of treatment, followed by one, two, three, four or five weeks off; (viii) every month. The cycle may be repeated 4,6, 8, 10, 12, 16 or 20 or more times.

Alternatively, the immunoconjugate may be administered as one dose every 2 or 3 weeks, repeating for a total of at least 3 doses. Alternatively, twice weekly for 4-6 weeks. If the dose is reduced to about 200-300mg/m ² (340 mg per dose for 1.7m patients), can be administered once or even twice a week for 4 to 10 weeks. Alternatively, the dosage regimen may be reduced, i.e. every 2 or 3 weeks for 2-3 months. However, it has been determined that even higher doses, such as 12mg/kg once a week or once every 2-3 weeks, can be administered by slow intravenous infusion for repeated dosing cycles. The dosage regimen may optionally be repeated at other intervals, and the dosages may be administered by various parenteral routes, with appropriate adjustment of the dosage and regimen.

In certain preferred embodiments, SN-38-conjugated anti-HLA-DR can be administered subcutaneously. For subcutaneous administration, the dosage of an ADC, such as IMMU-140(hL243-CL2A-SN-38), may be limited by the ability to concentrate the ADC without precipitation or aggregation, and the volume of administration that can be administered subcutaneously (preferably, 1,2, or 3ml or less). Thus, for subcutaneous administration, the ADC may be administered at 2 to 4mg/kg, daily for 1 week, or 3 times per week for 2 weeks, or twice per week for 2 weeks, followed by rest. Maintenance doses of ADC may be administered intravenously or subcutaneously every two to three weeks or monthly following induction. Alternatively, induction can be performed as follows: 2 to 4 cycles of intravenous administration at 8-10mg/kg (in two 21-day cycles, each cycle administering ADC on day 1 and 8 with a one week rest period in between) followed by subcutaneous administration as the active dose one or more times per week, or as maintenance therapy. The dose may be adjusted based on the provisional tumor scan and/or by analyzing Trop-2 positive circulating tumor cells.

In some preferred embodiments, the immunoconjugate is for use in the treatment of cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More specific examples of these cancers are described below, including: squamous cell cancer (e.g., epithelial squamous cell cancer), ewing's sarcoma, wilms' tumor, astrocytoma, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, hepatocellular cancer, neuroendocrine tumor, medullary thyroid cancer, differentiated thyroid cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, renal or renal cancer, prostate cancer, vulval cancer, anal cancer, penile cancer, and head and neck cancer. The term "cancer" includes primary malignant cells or tumors (e.g., those whose cells do not migrate to a site in the subject's body other than the site of the original malignant tumor or tumor) and secondary malignant cells or tumors (e.g., those resulting from metastasis, which migrate to a second site different from the site of the original tumor).

Other examples of cancer or malignancy include, but are not limited to: acute childhood lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myelogenous leukemia, adult hodgkin's lymphoma, adult lymphocytic leukemia, adult non-hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignancy, anal carcinoma, astrocytoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, brain stem glioma, brain tumor, breast carcinoma, renal pelvis and ureter carcinoma, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical carcinoma, child (primary) hepatocellular carcinoma, child (primary) liver cancer, pediatric (primary) carcinoma, neuroblastoma, renal carcinoma, primary carcinoma, neuroblastoma, colorectal carcinoma, and colorectal carcinoma, Childhood acute lymphoblastic leukemia, childhood acute myelogenous leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumors, childhood hodgkin's disease, childhood hodgkin's lymphoma, childhood hypothalamic and visual pathway gliomas, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-hodgkin's lymphoma, childhood pineal and supratentorial primitive neuroectodermal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine pancreatic islet cell carcinoma, endometrial cancer, ependymoma, epithelial cancer, esophageal cancer, ewing's sarcoma, and related tumors, Exocrine pancreatic cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher's Disease, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumor, germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hodgkin's lymphoma, hypergammaglobulinemia, hypopharynx cancer, intestinal cancer, intraocular melanoma, islet cell cancer, islet cell pancreatic cancer, kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liver cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic occult primary cervical squamous carcinoma, metastatic primary cervical carcinoma, metastatic squamous cell carcinoma, cervical carcinoma, malignant lymphoma, hemangioblastoma, neuroblastoma, hemangioblastoma, carcinoma of biliary tract, carcinoma of the head and mouth, thyroid, cervical carcinoma, prostatic carcinoma, cervical carcinoma of head and head carcinoma of head and head, Multiple myeloma, multiple myeloma/plasma cell tumor, myelodysplastic syndrome, myelogenous leukemia, myeloproliferative disorders, nasal and sinus cancers, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin's lymphoma, non-melanoma skin cancers, non-small cell lung cancer, occult primary metastatic squamous neck cancer, oropharyngeal cancer, osseous/malignant fibrosarcoma, bone/malignant fibrous histiocytoma, osteosarcoma/malignant fibrous histiocytoma of bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemia (pararoteinemia), polycythemia vera, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, cervical cell carcinoma, head, Renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, granulomatous sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous neck cancer, gastric cancer, supratentorial primary neuroectodermal and pineal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, renal pelvis and ureter transitional cell cancer, transitional renal pelvis and ureter cancer, trophoblastic tumors, ureter and renal pelvis cell cancer, urinary tract cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulval cancer, waldenstrom's macroglobulinemia, wilms' tumor, and any other hyperproliferative disease located in the above listed organ systems other than neoplasms.

The methods and compositions described and claimed herein are useful for treating malignant or pre-malignant conditions and preventing progression to tumors or malignant states, including but not limited to those conditions described above. Such use indicates a condition known or suspected of having previously developed a neoplastic neoplasm or cancer, particularly in the event of non-tumour cell growth arising from hyperplasia, metaplasia or most particularly dysplasia (for a review of such abnormal growth conditions see robblins and Angell, Basic Pathology, 2 nd edition, w.b. saunders co., philidelphia, pages 68-79 (1976)).

Dysplasia is often a precursor to cancer and is found primarily in epithelial cells. It is the most disorderly form of growth of non-tumor cells, involving loss of individual cellular uniformity and cellular structural orientation. Dysplasia characteristically occurs where there is chronic irritation or inflammation. Dysplastic disorders which may be treated include, but are not limited to, anhidrotic ectodermal dysplasia (anhidrotic ectodermal dysplasia), heteroplanar dysplasia (antrodial dysplasia), choking thoracic dysplasia (asphyxiating thoracogenic dysplasia), atrial-finger dysplasia (atriodigital dysplasia), bronchopulmonary dysplasia (bronopologic dysplasia), cerebral dysplasia (cerebral dysplasia), cervical dysplasia (cerial dysplasia), chondroaortic dysplasia (chondrodysplasia), clavicular cranial dysplasia (clavicular dysplasia), congenital ectodermal dysplasia, cranial dyslasia (cranial dysplasia), metatarsophalangeal dysplasia (dysphylogenia), carpal dysplasia (dysplasia), cranial dysplasia, cranial dyslasia (cranial dysplasia), cranial dyslasia (cerebrodia dyslasia), and cervical dysplasia (cerebralis dyslasia), and cervical dysplasia (cisgrade dyslasia), and cervical dysplasia (cisternaria, and cervical dysplasia, and cephalia (cisternaria, cephalia, and cephalia, and cephalia, and abnormal cephalia, or abnormal cephalia, or abnormal cephalia, hemiepiphyseal dysplasia (dysepiphyseal dysplasia), multiple epiphyseal dysplasia (dysepiphyseal dysplasia multiplex), punctate epiphyseal dysplasia (dysepiphyseal dysplasia pulata), epithelial dysplasia, facial dysplasia (dysarthritic dysphylia), familial dysplasia of the fibers of the jaw bone (familial dysarthria of the jaws), familial dysplasia white metaphyseae (familial white dysplasia), fibromyalgia dysplasia, fibrodysplasia, fibrous dysplasia of the bones (fibrous dysplasia of the bones), flourishing dysplasia (dysarthria dysphylia), hereditary dysplasia of the kidney-retina, perspirant dysplasia, dyshidrosis of the outer blastodermia (dysphylia), dyshidrosis of the outer blastophylia (dysphylia diaphyseal dysplasia), dyshidrosis of the lower jaw, dysphylogenia (dysphylogenia diaphyseal dysplasia), dysphylia of the lower jaw metaphysis (dysphylia), dysphylogenia diaphyseal dysplasia, dysphylogenia of the lower jaw bone (dysphylia), dysphylogenia of the outer dysplasia of the outer metaphysis of the lower metaphysis (dysphylogenia, diaphyseal dysplasia, and abnormal diaphyseal dysplasia, and abnormal diaphyseal dysplasia, or the upper metaphysis of the outer metaphysis of the upper metaphysis of the lateral metaphysis of the upper bone, Mondini dysplasia (Mondini dysplasia), single bone fibrodysplasia (synostosis), mucosal epithelial dysplasia (mucoepithelial dysplasia), multiple epiphyseal dysplasia (multiple epiphyseal dysplasia), auricular spondylotic dysplasia (oculoauricular dysplasia), ocular dental dysplasia (oculodetical dysplasia), ocular vertebral dysplasia (oculodermatitic dysplasia), dental dysplasia (odontogenic dysplasia), ocular submandibular dysplasia (oculomomanual dysplasia), periapical dental dysplasia (periapical dysplasia dysphylia), multi-bone fibrodysplasia (synostotic dysplasia), pseudodysplasia (synostotic dysplasia-synostosis), and spinal dysplasia (spinal-dysplasia).

Other pre-neoplastic diseases that may be treated include, but are not limited to, benign proliferative diseases (e.g., benign tumors, fibrocystic disorders, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia), vitiligo, keratosis (keratoses), Bowen's disease, Farmer's skin, solar cheilitis, and solar keratosis.

In some preferred embodiments, the methods of the invention are used to inhibit the growth, development and/or metastasis of cancer, particularly those listed above.

Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, the progression and/or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias; e.g., acute lymphocytic leukemia, acute myelogenous leukemia [ including myeloblasts, promyelocytes, myelomonocytes, monocytes, and erythroleukemia ]) and chronic leukemias (e.g., chronic myelogenous [ myelogenous ] leukemia and chronic lymphocytic leukemia); polycythemia vera (polycythemia vera), lymphomas (e.g., hodgkin's disease and non-hodgkin's disease), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma (lymphagionedothieiosarcoma), synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, and non-malignant tumors, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma (emangioblastoma), acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that can be treated with immunoconjugates can include acute and chronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, glandular syndrome, bullous pemphigoid, diabetes, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, Takayasu arteritis, ANCA-associated vasculitis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, thromboangiitis obliterans, sjogren syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, bullous pemphigomphiasis, rheumatic fever, and rheumatoid arthritis, Pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tuberculosis of the spinal cord, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis or fibro-alveolar inflammation.

Reagent kit

Various embodiments may be directed to kits comprising components suitable for treating diseased tissue in a patient. An exemplary kit may contain at least one conjugated antibody or other targeting moiety as described herein. If the composition containing the components for administration is not formulated for delivery through the digestive tract, for example by oral delivery, a device capable of delivering the kit components by some other route may be included. One type of device for applications such as parenteral delivery is a syringe for injecting a composition into a subject. Inhalation devices may also be used.

The kit components may be packaged together or divided into two or more containers. In some embodiments, the container may be a vial containing a sterile lyophilized formulation of the composition suitable for reconstitution. The kit may also contain one or more buffers suitable for reconstituting and/or diluting other reagents. Other containers may be used including, but not limited to, pouches, trays, boxes, tubes, and the like. The kit components may be aseptically packaged and maintained in containers. Another component that may be included is instructions for use by the person using the kit.

Examples

The following examples illustrate various embodiments of the present invention without limiting its scope.

SUMMARY

The abbreviations used below are: DCC, dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; EEDQ, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline; MMT, monomethoxytrityl; PABOH, p-aminobenzyl alcohol; PEG, polyethylene glycol; SMCC, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester; TBAF, tetrabutylammonium fluoride; TBDMS, tert-butyldimethylsilyl chloride.

Chloroformates of the hydroxy compounds in the following examples were prepared according to the method described by Moon et al (J. medicinal Chem.51:6916-6926,2008) using triphosgene and DMAP. Extractive work-up means extraction with chloroform, dichloromethane or ethyl acetate and optionally washing with saturated bicarbonate, water and saturated sodium chloride. Unless otherwise stated, flash chromatography was performed using 230-. Reverse phase HPLC was performed by method a using a 7.8 x 300mm C18HPLC column, equipped with a pre-column filter, and performed for 10 minutes at a flow rate of 3 mL/min using a solvent gradient from 100% solvent a to 100% solvent B, and held at 100% solvent B at a flow rate of 4.5 mL/min for 5 or 10 minutes; or by method B using a 4.6 x 30mm xbridge C18,2.5 μm column, equipped with a pre-column filter, and using a solvent gradient from 100% solvent a to 100% solvent B at a flow rate of 1.5 mL/min for 4 minutes, and 100% solvent B at a flow rate of 2 mL/min for 1 minute. Solvent a was 0.3% aqueous ammonium acetate, pH 4.46, and solvent B was 9:1 aqueous acetonitrile-ammonium acetate (0.3%), pH 4.46. HPLC was monitored by a dual in-line absorbance detector set at 360nm and 254 nm.

Example 1 anti-HLA-DR antibody drug conjugate IMMU-140(hL243-CL2A-SN-38) at HLA-DR ⁺ In vitro and in vivo efficacy in cancer

Relapsed AML (acute myeloid leukemia), ALL (acute lymphocytic leukemia), and MM (multiple myeloma) continue to be therapeutic challenges. IMMU-114(hL243) is a humanized anti-HLA-DR IgG ₄ Monoclonal antibodies engineered to lack effector cell function but retain HLA-DR binding and broad anti-tumor effects in a variety of hematological tumors (SteinR et al, blood.2010; 115: 5180-90). When given subcutaneously, it proved effective in initial phase I clinical trials of relapsed or refractory NHL and CLL with good safety (clinical trials. gov, NCT 01728207).

In vitro, AML has been shown to be resistant to the anti-tumor effects of IMMU-114 despite high expression levels of HLA-DR. Similarly, IMMU-114 exhibits a range of anti-tumor effects ranging from as low as 9% to as high as 69% in a variety of different human ALL, CLL and MM cell lines.

To improve the anti-tumor activity of IMMU-114, an antibody-drug conjugate (ADC) designated IMMU-140 was prepared in which IMMU-114 was conjugated to the active metabolite SN-38 of irinotecan. Another adc (sacituzumab govitecan) utilizing SN-38 studied in solid tumors has good tolerability with clinically significant objective responses in patients given over multiple cycles of more than 6 months, with controlled neutropenia being the major toxicity. Therefore, we aimed to determine if SN-38, a drug not commonly used in hematopoietic cancers, would prove to be an effective and safe therapeutic drug when targeted with IMMU-114 antibodies.

In this study, the in vitro and in vivo activity of hL243-SN-38(IMMU-140) versus the parent IMMU-114 was examined in human AML, ALL, MM and CLL xenografts.

Method

The use of SN-38 with hL243 IgG has been described previously ₄ Methods of conjugation, and results in drug to antibody ratios of 6.1 to 6.6(Moon SJ et al j. med. chem.2008; 51: 6916-26). The SN-38 linker (below, left) contains a short polyethylene glycol (PEG) moiety to impart water solubility. Maleimide groups were introduced to rapidly thiol-maleimide conjugate to the mildly reduced antibody. The benzyl carbonate site provides a pH mediated cleavage site to release the drug from the linker. A cross-linking linker is attached to the 20-hydroxyl position of SN-38 to keep the lactone ring of the drug from opening to the less active carboxylic acid form under physiological conditions. The structure of the ADC is shown in fig. 1.

The conjugate was characterized by size exclusion HPLC (not shown). Unmodified IMMU-114 and IMMU-140 conjugates with drug/antibody molar substitution of 6.1 were compared. Unmodified IMMU-114 was detected at 280nm, while the conjugate was detected at 360nm, the absorption wavelength of SN-38. Conjugate > 98% monomer (not shown).

Without evidence of any loss of binding specificity of the ADC, e.g. by cell-based ELISA, e.g. by IMComparable binding of MU-140 and IMMU-114 to HLA-DR positive human melanoma cell line (A-374) was demonstrated (FIG. 2). When associated with the b chain, both recognize the a chain. K _D The values are shown in table 2 below. Antibody control (h679) was a humanized anti-histamine-succinyl-glycine (HSG) IgG.

TABLE 2 binding affinities of IMMU-140 versus IMMU-114

	K D (nM)	95％C.I.	R 2
				IMMU-140	0.77	0.62 to 0.91	0.97
IMMU-114	0.65	0.53 to 0.77	0.97

For in vitro cytotoxicity assays, cells were seeded in 96-well plates (1 × 10) ⁴ Cells/well), then add each test agent: free SN-38 (2.5X 10) ^-7 To 3.8X 10 ^-12 M)、IMMU-140(2.5×10 ^-7 To 3.8X 10 ^-12 M, SN-38 equivalent) and IMMU-114 (4X 10) ^-8 To 6.2X 10 ^-13 M). Plates were incubated for 96 hours, then assayed by MTSAnd (5) determining the cell activity. Inhibition was determined as the percentage of viable cells in the treated wells compared to untreated controls.

Cells treated with IMMU-114 or IMMU-140 at a protein concentration of 10nM (2X 10) ⁶ ) Was assayed for apoptotic signaling, then lysed and the protein (25mg) resolved by SDS-PAGE. Proteins were transferred to PVD membranes for Western blotting.

For AML and MM disease models, NSG/SCID and C.B. -17SCID mice were injected intravenously with MOLM-14 (2X 10), respectively ⁶ ) Or CAG cells (1X 10) ⁷ ) The first 24 hours received 2Gy irradiation.

Intravenous injection of 1X 10 ⁷ ALL (MN-60) and CLL (JVM-3) were established in C.B-17SCID mice of cells.

All treatments were initiated 5 days after tumor cell injection. Test agents, including the non-targeted control SN-38-ADC, were administered twice weekly for 4 weeks at the doses shown in the figures (100 to 500 mg). Animals were sacrificed at disease progression characterized by the onset of hind limb paralysis or loss of more than 15% of body weight.

Results

HLA-DR expression was examined in a variety of human cancer cell lines. Human ALL, MM, CLL and AML cell lines were harvested from tissue cultures and analyzed for HLA-DR (alpha chain) expression by FACS using AlexaFluor-647 labeled IMMU-114. The Mean Fluorescence Intensity (MFI) of IMMU-114 and non-targeted control h679 antibody demonstrated high expression of HLA-DR in all four cell lines, with the MFI value of IMMU-114 being 6.58X 10, respectively ⁴ (MN-60 ALL cell line), 8.07X 10 ⁴ (CAG MM cell line), 7.87X 10 ⁴ (JVM-3CLL cell line) and 5.75X 10 ³ (MOLM-14AML cell line). In contrast, CAG, MN-60 and JVM-3 showed expression in comparison with AML cell line MOLM-14>Expression 10 times higher.

In vitro, IMMU-140 reached IC at low nM concentrations (0.8 to 7.1nM) in ALL four hematopoietic tumor types (ALL, MM, CLL and AML) ₅₀ Values, as shown in table 3 below. In vitro, JVM-3 showed the highest sensitivity to both IMMU-140 and IMMU-114. The remaining three cell lines showed only 50% or more growth in the presence of SN-38 and IMMU-140And (4) inhibiting. Although 50% inhibition was not achieved in those cell lines using IMMU-114, the unconjugated antibody mediated significant growth inhibition at 40nM in cag (mm) and MN-60(ALL) compared to the non-targeted control antibody. MOLM-14 was also resistant to IMMU-114, but sensitive to IMMU-140, using two additional AML cell lines (Stein R et al, blood.2010; 115:5180-90), as previously reported.

TABLE 3 in vitro cytotoxicity of IMMU-140 versus IMMU-114 in AML, ALL, CLL and MM cells.

IMMU-140 concentration shown as SN-38 equivalents

^* IC of free SN-38 compared to IMMU-140 in MOLM-14 ₅₀ Significant difference (P ═ 0.0266)

Significant inhibition compared to control antibody (P)<0.0061)

IMMU-140 demonstrates dual apoptotic signaling pathways mediated by anti-HLA-DR binding and delivery of SN-38 by its target cells. IMMU-114 signals apoptosis via p-ERK-1/2 and apoptosis-inducing factor (AIF) in NHL, ALL, MM and CLL, but not in AML (SteinR et al. blood.2010; 115(25): 5180-. Here we demonstrate that both IMMU-114 and IMMU-140 are able to mediate phosphorylation of ERK1/2 and up-regulate AIF in three different hematopoietic cell lines (not shown), including the AML cell line MOLM-14 (not shown), suggesting a defect in AML in other signaling components of the pathway, as it is insensitive to IMMU-114 both in vitro and in vivo.

Importantly, IMMU-140, via its SN-38 payload, also mediates PARP lysis in all three cell lines, including MOLM-14 (not shown). The resulting double stranded dna (dsdna) breaks were most evident in cells treated with IMMU-140 as evidenced by increased p-h2a.x levels (not shown).

In experimental MOLM-14AML, saline control and IMMU-114 treated mice died of rapid disease progression with Median Survival Time (MST) of only 14 days and 15 days, respectively (fig. 3). In contrast, survival increased more than 1.5-fold for mice treated with IMMU-140 (MST 37 days, P0.0031) (fig. 3). Furthermore, a reduction of the dose to 250mg IMMU-140 still provided a statistically significant improvement in survival rate of greater than 80% (MST 21 days, P0.0031) compared to saline and control ADC (anti-CEA-SN-38 IMMU-130) administered at the same dose (fig. 3).

In mice with MN-60ALL xenografts (figure 4), IMMU-114 provided > 60% improvement in survival compared to saline control (MST 37 days versus 22.5 days; P <0.0001), whereas IMMU-140 increased this by an additional 80% (MST 66.5 days), which was significantly better than ALL other treatments including IMMU-114 (P <0.0001) (figure 4). Treatment with IMMU-140 was well tolerated by mice without significant loss of body weight.

Mice with CAG MM xenografts (figure 5) had higher 151-day MSTs when treated with IMMU-140 compared to the saline control for 32 days (P < 0.0001). This survival benefit was also significantly higher than mice treated with bortezomib (0.89mg/kg) or control ADC + bortezomib (MST of both 32.5 days; P <0.0001) (fig. 5). Although not significant, IMMU-140 did provide > 60% improvement in survival compared to IMMU-114 treatment (MST 94.5 days, P0.0612) (fig. 5). Bortezomib treatment in combination with IMMU-114 or IMMU-140 did not improve survival above monotherapy (fig. 5).

Mice bearing JVM-3CLL xenografts (fig. 6) showed similar sensitivity to both IMMU-140 and IMMU-114. There was significant survival (P <0.0002) in mice treated with high (500mg) or low (100mg) dose of IMMU-140 compared to saline or control ADC treated mice (figure 6). Likewise, mice treated with either dose of IMMU-114 had >96 days of MST (P <0.0001 compared to saline and P <0.0003 compared to control ADC) (fig. 6). There was no significant difference between mice treated with IMMU-140 and IMMU-114 at the dose administered to the mice (figure 6). These results indicate that efficacy can be achieved at doses less than 100mg, indicating that IMMU-140 has a clinically broad therapeutic window in this disease.

In all experiments, treatment with IMMU-140 was well tolerated as evidenced by no significant loss of body weight.

Conclusion

IMMU-114 is an IgG devoid of immune function ₄ Mab, eliminates the known adverse events of previous HLA-DR mabs. HLA-DR recognized by IMMU-114 is expressed in a wide range of human hematopoietic and solid cancer types. Conjugation of 6-8 SN-38 molecules to IMMU-114(hL 243-SN-38) via a cleavable linker did not alter its binding to HLA-DR positive cells.

hL243-SN-38(IMMU-140) provides additional benefits of dual therapeutics through direct antitumor activity mediated by IMMU-114HLA-DR binding moieties (p-ERK1/2 and AIF signaling) and additional cytotoxic effects of SN-38 delivered to cells (caspase cascade and PARP cleavage). IMMU-140 antibody-drug conjugates showed preclinical higher efficacy in ALL and AML than naked IMMU-114, and increased (if not significant) survival benefit in experimental MM and CLL. Overall, the dual therapeutic potential of SN-38-conjugated IMMU-114(IMMU-140) allows for the ability to treat a variety of HLA-DR positive hematopoietic and solid cancers.

Treatment with IMMU-140ADC demonstrated superior efficacy to IMMU-114 (which is clinically active in NHL and CLL) in both AML and ALL xenografts, and was beneficial in MM and CLL. Most importantly, in IMMU-114 refractory AML, IMMU-140 showed significant anti-tumor effects without any undue toxicity. The data show that this novel ADC is useful for these refractory malignancies.

Example 2 IMMU-140 on HLA-DR ⁺ Efficacy in human melanoma

The expression of HLA-DR antigen is not limited to hematopoietic cancers, but also occurs in skin cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, endometrial cancerCervical cancer, testicular cancer, melanoma, renal cancer, and liver cancer. The study was aimed at examining IMMU-140 in nonhematopoietic HLA-DR ⁺ Efficacy in tumors.

Cell binding studies

Chemiluminescent substrate systems are used to detect antibodies that bind to cells. Briefly, A-375 human melanoma cells were seeded overnight in 96-black well flat-clear-bottom plates (black-well). The hL 243-gamma 4P antibody was added to triplicate wells (final concentration in wells 2. mu.g/mL). As a control for non-specific binding, humanized anti-CD 22 antibody was similarly added to another set of triplicate wells. Two plates were set up, one at Room Temperature (RT) and one at 4 ℃. After 1 hour of incubation, the medium was removed, the cells were washed with fresh cold medium, and then goat anti-human horseradish peroxidase conjugated secondary antibody diluted 1:20,000 was added and maintained at 4 ℃ for 1 hour. The plate was washed again and then added

And (3) a reagent.

Using ENVISION ^TM The microplate reader reads the luminescence of the plate. The average luminescence value was determined and plotted as shown in fig. 7. The average luminescence of hL 243. gamma.4 on A-375 cells was 48-fold background and 25-fold non-specific control at 4 ℃ and room temperature, consistent with high expression of HLA-DR on this human melanoma cell line. In summary, 4 of the 4 human melanoma cell lines tested (A-375, SK-MEL-28, SK-MEL-5 and SK-MEL-2) were positive for hL243- γ 4P binding (48-, 25-, 12-and 2-fold over background, respectively).

In vivo efficacy in A-375 tumor xenografts

Athymic NCr nu/nu nude mice were injected subcutaneously with 5X 10 ⁶ A-375 cells/mouse. Once the tumor size reached about 0.3cm ³ Animals were divided into 6 different treatment groups of 10 mice each. Mice received 250 μ g of intradermal IMMU-140(hL243-SN-38) (DAR ═ 5.05) twice a week for a duration of timeAnd (4) four weeks. The ADC control group consisted of mice that received the same dose of non-tumor targeting anti-CD 20ADC (hA20-CL 2A-SN-38; DAR ═ 6.08) in the same protocol. In addition, one group of mice received naked hL243- γ 4P alone (250 μ g), and one group received hL243- γ 4P plus irinotecan (250 μ g MAb +7.5 μ g irinotecan) at a dose equal to the ADC dose. The last group received irinotecan only at a dose 10-fold higher than the amount of SN-38 carried by hL243-SN-38 (i.e., 75 μ g). All irinotecan injections were administered intravenously. The last group of mice received saline only (100 μ L intraperitoneal injection). The treatment groups are summarized in table 4 below.

TABLE 4 treatment group of nude mice with melanoma

Tumors were measured and mice weighed weekly. If the tumor volume size exceeds 2.0cm ³ The animals are sacrificed for disease progression. Partial response is defined as the shrinkage of the tumor from the initial size>30 percent. The disease is stable when the tumor volume remains between 70% and 120% of the initial size. The time To Tumor Progression (TTP) was determined as the time for the tumor to grow more than 20% from its nadir.

Statistical analysis of tumor growth data was based on area under the curve (AUC) and TTP. A profile of individual tumor growth was obtained by linear curve modeling. Prior to statistical analysis of growth curves, an F-test was used to determine the equality of variance between groups. The two-tailed t-test was used to assess statistical significance between all individual treatment groups and controls, except for the saline control group, where a single-tailed t-test was used in the analysis. Due to the incompleteness of some growth curves (due to death), statistical comparisons of AUC were only made up to the time the first animal in the group was sacrificed. TTP values between the comparison groups were compared using a two-tailed t-test.

Mean tumors in all groups at the start of treatmentThe volume is 0.314 +/-0.078 cm ³ . The mean tumor growth curve is shown in figure 8. This disease model proved to be very aggressive, with saline control tumors progressing rapidly (TTP 7 days; table 5). Although tumors progressed equally in the control group, all treatments were able to slow tumor growth relative to the saline control (P)<0.0142, AUC) (fig. 8 and table 5). However, mice treated with hL243-SN-38 alone showed significant anti-tumor effects (P) compared to all other groups<0.0244; AUC) (fig. 8 and table 5). All mice in this group were partial responders, two of which had no tumor at the end of the experiment on day 70 of treatment. This resulted in a more than 3-fold delay in tumor progression (P) compared to all other non-ADC control groups<0.0005) (fig. 8 and table 5). Although this tumor was sensitive to non-specific ADC, treatment with hL243-SN-38 delayed TTP by more than 80% (28 ± 9.9 days versus 15.6 ± 7.7 days; P ═ 0.012, respectively) compared to mice treated with control ADC (fig. 8 and table 5). These data indicate that treatment with hL243-SN-38 results in significant tumor regression and delay in disease progression even in a mouse disease model of aggressive human melanoma tumors (fig. 8 and table 5).

Example 3 preparation of CL2A-SN-38

To a mixture of commercially available Fmoc-Lys (MMT) -OH (0.943g), p-aminobenzyl alcohol (0.190g) in dichloromethane (10mL) was added EEDQ (0.382g) at room temperature and stirred for 4 hours. Extractive workup followed by flash chromatography gave 1.051g of a white foamy substance. All HPLC analyses were performed by method B as described in 'overview' section 0148. HPLC retention time: at 3.53 minutes, electrospray mass spectra were shown at M/e 745.8(M + H) and M/e 780.3(M + Cl) ^- ) Peaks at (b), consistent with the structure. This intermediate (0.93g) was dissolved in diethylamine (10mL) and stirred for 2 hours. After removal of the solvent, the residue was washed in hexane to obtain 0.6g of intermediate (2 in scheme 3) as a colorless precipitate (HPLC purity 91.6%). HPLC retention time: 2.06 minutes. Electrospray mass spectrometry displayPeaks at M/e 523.8(M + H), M/e 546.2(M + Na) and M/e 522.5 (M-H).

The crude intermediate (0.565g) was coupled with commercially available O- (2-azidoethyl) -O ' - (N-diethanolyl-2-aminoethyl) heptaethylene glycol (' PEG-N3 ', 0.627g) using EEDQ in dichloromethane (10 mL). Solvent removal and flash chromatography gave 0.99g of product (3 in scheme 3; light yellow oil; 87% yield). HPLC retention time: 2.45 minutes. Electrospray mass spectroscopy showed peaks at M/e 1061.3(M + H), M/e 1082.7(M + Na), and M/e 1058.8(M-H), consistent with the structure. This intermediate (0.92g) was reacted with 10-O-TBDMS-SN-38-20-O-chloroformate in dichloromethane (10mL) under argon for 10 minutes. The mixture was purified by flash chromatography to give 0.944g of a pale yellow oil (6 in scheme 3; yield ═ 68%). HPLC retention time: 4.18 minutes. To a solution of this intermediate (0.94g) in dichloromethane (10mL) was added a mixture of TBAF (1M in THF, 0.885mL) and acetic acid (0.085mL) in dichloromethane (3mL), followed by stirring for 10 min. The mixture was diluted with dichloromethane (100mL) and washed with 0.25M sodium citrate and brine. The solvent was removed to give 0.835g of a yellow oily product. HPLC retention time: 2.80 minutes, (99% purity). Electrospray mass spectroscopy showed peaks at M/e 1478(M + H), M/e 1500.6(M + Na), M/e 1476.5(M-H), M/e 1590.5(M + TFA), consistent with the structure.

Scheme 3: preparation of CL2A-SN-38

The azido-derivatized SN-38 intermediate (0.803g) was reacted with 4- (N-maleimidomethyl) -N- (2-propynyl) cyclohexane-1-carboxamide (0.233g) in dichloromethane (10mL) in the presence of CuBr (0.0083g), DIEA (0.01mL) and triphenylphosphine (0.015g) for 18 hours. Extractive workup, including washing with 0.1M EDTA (10mL), flash chromatography gave 0.891g of yellow foam (yield 93%), HPLC retention time: 2.60 minutes. Electrospray mass spectroscopy showed peaks at M/e 1753.3(M + H), M/e 1751.6(M-H), 1864.5(M + TFA), consistent with the structure. Finally, deprotection of the penultimate intermediate (0.22g) with a mixture of dichloroacetic acid (0.3mL) and anisole (0.03mL) in dichloromethane (3mL) followed by ether precipitation gave 0.18g (97% yield) of CL 2A-SN-38; (7) in scheme 3, as a pale yellow powder. HPLC retention time: 1.88 minutes. Electrospray mass spectroscopy showed peaks at M/e 1480.7(M + H), 1478.5(M-H), consistent with the structure.

Example 4 conjugation of bifunctional SN-38 products to lightly reduced antibodies

Each antibody was reduced with Dithiothreitol (DTT) in a 50-70 fold molar excess for 45 minutes at 37 ℃ (bath) in 40mM PBS (pH 7.4) containing 5.4mM EDTA. The reduced product was purified by size exclusion chromatography and/or diafiltration and buffer exchanged to a suitable buffer at pH 6.5. The thiol content was determined by the Ellman assay and ranged from 6.5 to 8.5 SH/IgG. Alternatively, the antibody is reduced with tris (2-carboxyethyl) phosphine (TCEP) in phosphate buffer at pH 5-7 and then conjugated in situ. The reduced MAb was reacted with CL2A-SN-38 using 7-15% v/v DMSO as a co-solvent and incubated for 20 minutes at ambient temperature. The conjugate was purified by centrifugation SEC, through a hydrophobic column, and finally by ultrafiltration-diafiltration. The SN-38 of the product was determined by absorbance at 366nm and correlated with a standard value, while the protein concentration was inferred from the absorbance at 280nm, correcting for the overflow of SN-38 absorbance at this wavelength. Thus, the SN-38/MAb substitution ratio was determined. The purified conjugate was stored as a lyophilized formulation in a glass vial, capped under vacuum and stored in a-20 ℃ freezer. SN-38 Molar Substitution Ratios (MSRs) were obtained for some of these conjugates, which were typically 5-7, shown in Table 6.

TABLE 6 SN-38/MAb Molar Substitution Ratio (MSR) in some conjugates

MAb	Conjugates	MSR
			hMN-14	hMN-14-[CL2A-SN-38]	6.1
hRS7	hRS7-CL2A-SN-38, using the drug linker of example 10	5.8
			hA20	hA20-CL2A-SN-38, using the drug linker of example 10	5.8
hLL2	hLL2-CL2A-SN-38, using the drug linker of example 10	5.7
			hPAM4	hPAM4-CL2A-SN-38 using the drug linker of example 10	5.9

Example 5 treatment of refractory metastatic colon cancer (mCRC) with hL243-SN-38

The patient was a 67 year old male with metastatic colon cancer. After a transverse colectomy shortly after diagnosis, the patient then received 4 cycles of FOLFOX chemotherapy in the context of neoadjuvant therapy, followed by a partial hepatectomy to remove metastatic lesions in the left lobe of the liver. The following is the auxiliary FOLFOX protocol, for a total of 10 FOLFOX cycles.

CT showed metastasis to the liver. His target lesion was a 3.0cm tumor of the left lobe of the liver. Non-target lesions include several low-attenuating masses in the liver. The baseline CEA was 685 ng/mL.

hL243-SN-38(10mg/kg) was administered every other week for 4 months after the patient signed an informed consent. Patients experienced nausea (grade 2) and fatigue (grade 2) after the first treatment and continued treatment without major adverse events. Completed first response assessment (after 8 doses) showed that the target lesion contracted 26% by Computed Tomography (CT) and his CEA level was reduced to 245 ng/mL. In the second response assessment (after 12 doses), the target lesion was reduced by 35%. His overall health and clinical symptoms were significantly improved.

Example 6 treatment of relapsed follicular lymphoma with IMMU-140 (anti-HLA-DR-SN-38)

This 68 year old male trial drug, IMMU-114-SN-38 (anti-HLA-DR-SN-38), was given a dose of 10mg/kg weekly for 3 weeks, followed by a second course of 3 additional weeks after receiving R-CHOP chemotherapy for follicular lymphoma manifested as widespread disease and bone marrow involvement in multiple regional lymph nodes (cervix, axilla, mediastinum, groin, abdomen). His changes in the index tumor lesions were then assessed by CT and showed a 23% reduction according to the CHESON criteria. This treatment was repeated for 2 additional courses and then showed a 55% reduction in tumor by CT, which is a partial response.

Example 7 treatment of relapsed Chronic lymphocytic leukemia with IMMU-140

67 year old men with a history of CLL showed disease recurrence following previous treatment with fludarabine, dexamethasone and rituximab and CVP regimen, as defined by the international seminal lymphocyte leukemia seminar and the world health organization classification. He now has fever and night sweats associated with generalized lymphadenectasis, reduced hemoglobin and platelet production, and a rapid rise in white blood cell counts. His LDH was elevated and beta-2-microglobulin was almost twice as normal. The patient was given treatment with the IMMU-114-SN-38 conjugate at a dosing schedule of 8mg/kg per week for 4 weeks, rested for 2 weeks, and the cycle was repeated again. The evaluation showed that the patient's hematological parameters improved and his circulating CLL cell number appeared to be reduced. Treatment was then resumed for another 3 cycles, after which his haematological and laboratory values indicated that he had a partial response.

Example 8 immunoconjugates storage

The above conjugate was purified and buffer exchanged with 2- (N-morpholino) ethanesulfonic acid (MES) (pH 6.5) and further formulated with trehalose (25mM final concentration) and polysorbate 80 (0.01% v/v final concentration), the final buffer concentration becoming 22.25mM upon addition of excipients. The formulated conjugate was lyophilized and stored in a sealed vial at 2-8 ℃. The lyophilized immunoconjugate is stable under storage conditions. Maintaining its physiological activity.

***

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions without undue experimentation. All patents, patent applications, and publications cited herein are incorporated by reference.

Claims (24)

1. Use of immunoconjugates for the preparation of medicaments for the treatment of HLA-DR ⁺ Use in medicine of cancer, comprising administering to a patient suffering from HLA-DR ⁺ Administration of an immunoconjugate comprising SN-38 conjugated to an anti-HLA-DR antibody or antigen-binding fragment thereof to a human patient with cancer, wherein the HLA-DR antibody ⁺ The cancer is acute myeloid leukemia, wherein the anti-HLA-DR antibody or antigen binding fragment thereof comprises: a heavy chain CDR1 consisting of the sequence shown in SEQ ID NO. 1, a heavy chain CDR2 consisting of the sequence shown in SEQ ID NO. 2, and a heavy chain CDR3 consisting of the sequence shown in SEQ ID NO. 3, and a light chain CDR1 consisting of the sequence shown in SEQ ID NO. 4, a light chain CDR2 consisting of the sequence shown in SEQ ID NO. 5, and a light chain CDR3 consisting of the sequence shown in SEQ ID NO. 6, and wherein the immunoconjugate has the structure MAb-CL2A-SN-38

2. The use of claim 1, wherein the anti-HLA-DR antibody is an hL243 antibody.

3. The use of claim 1 or 2, wherein the HLA-DR ⁺ The cancer does not respond to treatment with unconjugated anti-HLA-DR antibodies.

4. The use of claim 1 or 2, wherein the immunoconjugate is administered as first line therapy to a patient who has not previously received treatment for the cancer.

5. The use of claim 1, wherein the immunoconjugate is administered to a patient who has previously relapsed from or who has been resistant to at least one anti-cancer therapy.

6. The use of claim 1, wherein the immunoconjugate is administered to a patient unsuitable for stem cell or bone marrow transplantation.

7. The use of claim 1, wherein the immunoconjugate is administered at a dose of between 3mg/kg and 18 mg/kg.

8. The use of claim 7, wherein the dose is selected from 4mg/kg, 6mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 12mg/kg, 16mg/kg and 18 mg/kg.

9. The use of claim 1, wherein the immunoconjugate is administered at a dose of between 6mg/kg and 12 mg/kg.

10. The use of claim 1, wherein the immunoconjugate is administered at a dose of between 8mg/kg and 10 mg/kg.

11. The use of claim 1, wherein the cancer is metastatic.

12. The use of claim 5, wherein the patient is not responsive to treatment with irinotecan prior to treatment with the immunoconjugate.

13. The use as claimed in claim 1, wherein the 10-hydroxy position of SN-38 in MAb-CL2A-SN-38 is a 10-O-ester or a 10-O-carbonate derivative using a ' COR ' moiety, where ' CO ' is a carbonyl group and the ' R ' group is selected from (i) N, N-disubstituted aminoalkyl ' N (CH) ₃ ) ₂ -(CH ₂ ) _n - ", wherein n is 1 to 10, and wherein the terminal amino group is optionally in the form of a quaternary salt; (ii) alkyl residue "CH ₃ -(CH ₂ ) _n -, where n is 0 to 10; (iii) alkoxy moiety "CH ₃ -(CH ₂ ) n-O- ", wherein n is 0-10; (iv) "N (CH) ₃ ) ₂ -(CH ₂ ) _n -O- ", wherein n is 2-10; or (v) "R ₁ O-(CH ₂ -CH ₂ -O) _n -CH ₂ -CH ₂ -O- ", wherein R ₁ Is ethyl or methyl, n is an integer having a value of from 0 to 10.

14. The use of claim 1, wherein 6 or more SN-38 molecules are attached per antibody molecule.

15. The use of claim 1, wherein the immunoconjugate comprises 7 to 8 SN-38 molecules conjugated to the antibody or antigen-binding fragment thereof.

16. The use of claim 1, wherein the antibody is an IgG1 or IgG4 antibody.

17. The use of claim 1, wherein the antibody is an IgG4 antibody.

18. The use of claim 1, wherein the antibody has an isotype selected from: g1m3, G1m3,1, G1m3,2, G1m3,1,2, nG1m1, nG1m1,2 and Km3 allotypes.

19. The use of claim 1, wherein the immunoconjugate dose is administered to the human patient once or twice per week in a regimen having cycles selected from: (i) weekly; (ii) every other week; (iii) for one week, then stop for two, three or four weeks; (iv) two weeks of treatment, followed by one, two, three or four weeks off; (v) for three weeks, then stop for one, two, three, four or five weeks; (vi) for four weeks, then discontinue for one, two, three, four or five weeks; (vii) treatment is for five weeks, then discontinued for one, two, three, four or five weeks; and (viii) monthly.

20. The use of claim 19, wherein the cycle is repeated 4,6, 8, 10, 12, 16 or 20 times.

21. The use of claim 1, wherein the anti-HLA-DR-SN-38 immunoconjugate is administered subcutaneously.

22. The use of claim 21, wherein the immunoconjugate is administered at a dose of 2 to 4 mg/kg.

23. The use of claim 21, wherein the immunoconjugate is administered in a volume of 1,2 or 3ml or less.

24. The use of claim 21, wherein a maintenance dose of the immunoconjugate is administered subcutaneously after intravenous administration of the immunoconjugate to the same subject.

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