RU2757395C2 - Therapy of metastatic urothelial cancer using antibody-drug conjugate satsituzumab govitecan (immu-132) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method for the treatment of metastatic urothelial cancer, including the administration to a human patient with urothelial cancer of an antibody-drug conjugate (hereinafter – ADC) containing SN-38 conjugated with an anti-Trop-2 antibody hRS7 or its antigen-binding fragment, wherein the specified ADC is administered at a dose of 8 mg/kg or 10 mg/kg, where the specified patient relapsed after one or more previous standard therapy and therapy with checkpoint inhibitors, or was refractory to one or more previous standard therapy and therapy with checkpoint inhibitors before treatment with the specified ADC.

EFFECT: aim of the invention is development of a method for the treatment of urothelial cancer.

17 cl, 9 dwg, 8 tbl, 6 ex

Description

RELATED APPLICATIONS

[01] This application is a partial continuation of US patent application No. 15/069208, filed 03/14/2016 (3/14/16), which was a partial continuation of US patent application No. 14/667982 (currently issued US patent No. 9493573) filed 03/25/2015, which was separated from U.S. Application No. 13/948732 (currently U.S. Patent No. 9028833), issued 7/23/2013, claiming priority under 35 U.S.C. Section 119 (e) Provisional U.S. Patent Applications 61/736684, filed 12/13/2012, and 61/749548, filed 01/07/2013. 15/069208 claims priority pursuant to 35 U.S.C. Section 119 (e), Provisional U.S. Patent Applications 62/133654, filed 03/16/2015, 62/133729, filed 03/16/2015, 62/138092, filed 03/25 2015, 62/156608, filed 05/04/2015, and 62/241881, filed 10/15/2015. This application claims, pursuant to section 119 (e) of Title 35, United States Code, to the priority of interim US patent application 62/428655, filed 12/01/2016. The text of each of the priority applications is incorporated herein in its entirety by reference.

LIST OF SEQUENCES

[02] This application contains the Sequence Listing, which was filed in ASCII format via the EFS-Web system and is hereby incorporated by reference in its entirety. The specified ASCII document, created on November 13, 2017, is named IMM356WO2_SL.txt and is 7835 bytes in size.

FIELD OF TECHNOLOGY

[03] This invention relates to the therapeutic use of immunoconjugates of antibodies or antigen-binding fragments of antibodies and camptothecins, such as SN-38, with improved ability to target various cancer cells in humans. In preferred embodiments, the antibodies and therapeutic fragments are linked by an intracellular cleavable bond that enhances therapeutic efficacy. In more preferred embodiments, the immunoconjugates are administered at specific dosages and / or at specific administration schedules that optimize the therapeutic effect. Optimized dosage and administration schedules of SN-38 conjugated antibodies in human therapeutic applications as disclosed herein have demonstrated unexpectedly improved efficacy that could not be predicted from animal model studies to effectively treat cancers resistant to standard cancer therapies. including irinotecan (CPT-11), the parent compound for SN-38. More preferably, the methods and compositions relate to use for the treatment of Trop2-positive cancer, in particular urothelial cancer, using the anti-Trop-2 hRS7-SN-38 immunoconjugate. In specific embodiments, the immunoconjugate can be administered to a human with Trop2-positive cancer at a dose of 3 to 18 mg / kg, more preferably 4 to 12 mg / kg, more preferably 8 to 10 mg / kg. In other preferred embodiments, the methods and compositions can be used to treat Trop2-positive cancer that recurs or is refractory to other standard cancer therapies, such as chemotherapy drugs. Surprisingly, anti-Trop-2-SN38 antibody-drug conjugates (ADCs) have been shown to be effective in the treatment of Trop2-positive cancers in patients who recur or show resistance to conventional anti-cancer agents, including irinotecan. In other preferred embodiments, anti-Trop-2-SN-38 CAP, such as IMMU-132, can be administered in combination with one or more other therapeutic agents that may synergize with CAP, such as microtubule inhibitors, PARP (poly (ADP-ribose) polymerase) inhibitors, Bruton's kinase inhibitors, or PI3K (phosphatidylinositol-3-kinase) inhibitors.

LEVEL OF TECHNOLOGY

[04] For many years, the goal of specific drug therapy scientists has been to use monoclonal antibodies (MAT) for the specific delivery of toxic agents in human cancer. Conjugates of tumor-associated MABs and suitable toxic agents have been developed, but have demonstrated variable success in cancer therapy in humans. The toxic agent is most often a chemotherapeutic drug, although particle-emitting radionuclides or bacterial or plant toxins have also been conjugated to MAT, especially for cancer therapy (Sharkey and Goldenberg, CA Cancer J Clin. 2006 Jul-Aug; 56 (4 ): 226-243).

[05] The advantages of using MAT-chemotherapeutic drug conjugates are that (a) the chemotherapeutic drug itself is structurally well defined; (b) the chemotherapeutic drug binds to the MAT protein using very well defined conjugation chemistry, often at specific sites distant from the antigen binding regions of the MAT; (c) greater reproducibility and generally less immunogenicity of MAT-chemotherapeutic drug conjugates can be achieved as compared to chemical conjugates comprising MAT and bacterial or plant toxins and are therefore more suitable for commercial development and regulatory approval; and (d) MAT-chemotherapeutic drug conjugates have several orders of magnitude lower systemic toxicity than radionuclide-MAT conjugates, especially with respect to radiation-sensitive bone marrow.

[06] Camptothecin (CPT) and its derivatives are a class of potent antineoplastic agents. Irinotecan (also referred to as CPT-11) and topotecan are CPT analogs that have been approved for the treatment of cancer (Iyer and Ratain, Cancer Chemother. Phamacol. 42: S31-S43 (1998)). Camptothecins act by inhibiting the enzyme topoisomerase I by stabilizing the topoisomerase I-DNA complex (Liu, et al. In The Camptothecins: Unfolding Their Anticancer Potential, Liehr JG, Giovanella, BC and Verschraegen (eds), NY Acad Sci., NY 922: 1 -10 (2000)). Camptothecins pose specific problems in the preparation of conjugates. One of the problems is the insolubility of most CPT derivatives in aqueous buffers. Second, CPT poses specific problems in structural modification for conjugation to macromolecules. For example, CPT itself contains only a tertiary hydroxyl group in the E ring. The hydroxyl functional group in the case of CPT must be linked to a linker suitable for subsequent protein conjugation; and in potent CPT derivatives such as SN-38, an active metabolite of chemotherapeutic CPT-11 and other C-10 hydroxyl derivatives such as topotecan and 10-hydroxy-CPT, the presence of phenolic hydroxyl at the C-10 position complicates the necessary derivatization of C- 20-hydroxyl. Third, the lability of the δ-lactone fragment of the E-ring of camptothecins under physiological conditions leads to a significant decrease in antitumor activity. Therefore, the conjugation protocol is performed in such a way that it is carried out at a pH of 7 or lower, in order to avoid opening of the lactone ring. However, a pH of 8 or higher is usually required to conjugate a bifunctional CPT having an amine-reactive group such as an active ester. Fourth, the intracellularly cleavable fragment is preferably included in a linker / spacer connecting CPT and antibodies or other binding fragments.

[07] There is a need for more efficient methods of making and administering antibody-CPT conjugates, such as antibody-SN-38 conjugates. Preferably, the methods include optimized dosing and administration regimens that maximize efficacy and minimize the toxicity of antibody-CPT conjugates in human therapeutic use.

SUMMARY OF THE INVENTION

[08] As used herein, the abbreviation "CPT" may refer to camptothecin or any of its derivatives, such as SN-38, unless expressly indicated otherwise. This invention addresses the unmet need in the art by providing improved methods and compositions for preparing and administering CPT-antibody immunoconjugates. Preferably, camptothecin is SN-38. The disclosed methods and compositions are suitable for use in the treatment of various diseases and conditions that are resistant or less sensitive to other forms of therapy, and may include diseases against which suitable antibodies or antigen-binding antibody fragments for selective targeting may be developed, or are available or known. ... Preferred diseases or conditions that can be treated with the immunoconjugates of this invention include Trop2-positive cancers such as metastatic urothelial cancer.

[09] Preferably, the targeting fragment is an antibody, an antibody fragment, a bispecific or other multivalent antibody, or another antibody molecule or compound. The antibody can be of different isotypes, preferably human IgG1, IgG2, IgG3 or IgG4, more preferably it contains human IgG1 hinge and constant region sequences. The antibody or fragment thereof can be a chimeric human-mouse antibody, a chimeric human-primate antibody, humanized (human framework and mouse hypervariable regions (CDRs)) or fully human antibodies, as well as variants thereof such as semi-IgG4 antibodies (called "(Unibodies)) as described by van der Neut Kolfschoten et al. (Science 2007; 317: 1554-1557). More preferably, the antibody or a fragment thereof can be designed or selected to contain human constant region sequences belonging to particular allotypes, which can result in decreased immunogenicity when the immunoconjugate is administered to a human. Preferred allotypes for introduction include non-G1m1 (nG1m1) allotypes such as G1m3, G1m3,1, G1m3,2, or G1m3,1,2. More preferably, the allotype is selected from the group consisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

[010] Suitable antibodies can bind to any disease associated antigen known to those skilled in the art. In cases where the disease state is cancer, for example, many antigens expressed or otherwise associated with tumor cells are known to those skilled in the art, including but not limited to carbonic anhydrase IX, alpha-fetoprotein (AFP), α-actinin-4, A3, antigen specific to antibody A33, ART-4, B7, Ba 733, BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon cancer specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, Trop-2, EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, H M1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-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), KC4 antigen, KS-1 antigen, KS1-4, Le-Y , LDR / FUT, macrophage migration inhibiting factor (MIF), MAGE, MAGE-3, MART-1, MART-2, 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, placental growth factor , p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG -72, tenascin, TRAIL receptors, TNF-α, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, factors complement C3, C3a, C3b, C5a, C5, marker of angiogenesis, bcl-2, bcl-6, Kras, oncogene marker and oncogene product (see, for example, Sensi et al., Clin Cancer Res 2006, 12: 5023-32 ; Parmiani et al., J Immunol 2007,178: 1975-79; Novellino et al. Cancer Immunol Immunother 2005,54: 187-207). Preferably, the antibody binds to CEACAM5, CEACAM6, Trop-2, AFP, MUC5ac, CD74, CD19, CD20, CD22, or HLA-DR. Most preferably, the antibody binds to Trop-2.

[011] Typical examples of anti-cancer antibodies that can be used include, without limitation, hR1 (anti-IGF-1R, US patent No. 9441043), hPAM4 (anti-MUC-5ac, US patent No. 7282567), hA20 (anti- CD20, US patent No. 7151164), hA19 (anti-CD19, US patent No. 7109304), hIMMU31 (anti-AFP, US patent No. 7300655), hLL1 (anti-CD74, US patent No. 7312318), hLL2 (anti-CD22, US patent No. 5789554), hMu-9 (anti-CSAp, US patent No. 7387772), hL243 (anti-HLA-DR, US patent No. 7612180), hMN-14 (anti-CEACAM5, US patent No. 6676924), hMN- 15 (anti-CEACAM6, US patent No. 8287865), hRS7 (anti-Trop-2, US patent No. 7238785), hMN-3 (anti-CEACAM6, US patent No. 7541440), Ab124 and Ab125 (anti-CXCR4, US patent No. 7138496), and the Examples sections of each of these patents or applications are incorporated herein by reference. More preferably, the antibody is IMMU-31 (anti-AFP), hRS7 (anti-Trop-2), hMN-14 (anti-CEACAM5), hMN-3 (anti-CEACAM6), hMN-15 (anti-CEACAM6) , hLL1 (anti-CD74), hLL2 (anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20 (anti-CD20). In a particularly preferred embodiment, the antibody is hRS7 (anti-Trop-2).

[012] Alternative antibodies used include, without limitation, abciximab (against glycoprotein IIb / IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab ( anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (against PD-1 receptor), nivolumab (against PD-1 receptor) , ipilimumab (anti-CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (against the IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20) , CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, US patent application 11/983372, deposited as ATCC PTA-4405 and PTA-4406), D2 / B (anti-PSMA, WO 2009/130575), tocilizumab (against IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab (anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (against CD3 receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE); anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes 45: 881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (Thermo Scientific, Rockford, IL), infliximab (Centocor, Malvern, PA), certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels, Belgium), adalimumab (Abbott, Abbott Park, IL), benlist (Human Genome Sciences); antibodies for the therapy of Alzheimer's disease such as Alz 50 (Ksiezak-Reding et al., 1987, J Biol Chem 263: 7943-47), gantenerumab, solanezumab and infliximab; antifibrin antibodies such as 59D8, T2G1s, MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202 (Celgene), HuMax-CD38 (Genmab), or daratumumab (Johnson & Johnson).

[013] In a preferred embodiment, the chemotherapeutic moiety is selected from camptothecin (CPT) and its analogs and derivatives, and more preferably it is SN-38. However, other chemotherapeutic agents that can be used include taxanes (e.g. baccatin III, taxol), epothilones, anthracyclines (e.g. doxorubicin (DOX), epirubicin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholineodoxorubicin-DOX), 2 β-pyrrolinodoxorubicin (2-PDOX) or a prodrug 2-PDOX (pro-2-PDOX); see, for example, Priebe W (ed.), ACS symposium series 574, published by American Chemical Society, Washington DC, 1995 (332pp ), and Nagy et al., Proc. Natl. Acad. Sci. USA 93: 2464-2469, 1996), benzoquinoid ansamycins exemplified by geldanamycin (DeBoer et al., Journal of Antibiotics 23: 442-447, 1970; Neckers et al., Invest. New Drugs 17: 361-373, 1999), and the like. Preferably, the antibody or fragment thereof binds to at least one chemotherapeutic fragment; preferably 1 to about 5 chemotherapeutic moieties; more preferably 6 or more chemotherapeutic moieties, more preferably about 6 to about 12 chemotherapeutic moieties.

[014] An example of a water-soluble derivative of CPT is CPT-11. A large body of clinical data is available regarding the pharmacology of CPT-11 and its in vivo conversion to active SN-38 (Iyer and Ratain, Cancer Chemother Pharmacol. 42: S31-43 (1998); Mathijssen et al., Clin Cancer Res. 7: 2182 -2194 (2002); Rivory, Ann NY Acad Sci. 922: 205-215, 2000)). The active form of SN-38 is about 2-3 orders of magnitude more potent than CPT-11. In certain preferred embodiments, the immunoconjugate can be the conjugate 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, hLL1-SN-38 or hLL2-SN-38.

[015] Various embodiments may relate to the use of the methods and compositions of this invention to treat cancer, including, but not limited to, non-Hodgkin's lymphomas, 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, acute lymphocytic leukemia, chronic lymphocytic leukemia, T-cell lymphomas and leukemias, multiple myeloma, Waldenstrom's macroglobulinemia, carcinomas, melanomas, sarcomas, gliomas, types of bone and skin cancers. Carcinomas can include carcinomas of the mouth, esophagus, gastrointestinal tract, pulmonary tract, lung, stomach, colon, breast, ovary, prostate, uterus, endometrium, cervix, bladder, pancreas, bone, brain, connective tissue , liver, gallbladder, bladder, kidney, skin, central nervous system and testicles. Preferably, the cancer is urothelial cancer, more preferably metastatic urothelial cancer, most preferably metastatic urothelial cancer that recurs or is refractory to standard cancer therapy such as chemotherapy.

[016] In certain embodiments related to cancer treatment, CAP can be used in combination with surgery, radiation therapy, chemotherapy, naked antibody immunotherapy, radioimmunotherapy, immunomodulators, vaccines, and the like. Such combination therapies can allow for the use of lower doses of each therapeutic agent in such combinations, thereby reducing some of the severe side effects and potentially reducing the required courses of therapy. In the absence or with minimal overlapping toxicity, full doses of each drug can also be administered. Surprisingly, combination therapy with antibody-SN38 immunoconjugates and microtubule inhibitors or PARP (poly (ADP-ribose) polymerase) inhibitors showed unexpected synergistic effects.

[017] The preferred optimal dosage of CAP may include a dose of 3 mg / kg to 18 mg / kg, which is preferably administered weekly, twice a week, or every two weeks. An optimal dosing schedule may include treatment cycles of two consecutive weeks of therapy followed by one, two, three or four weeks of rest, or alternating weeks of therapy and rest, or one week of therapy followed by two, three or four weeks of rest. or three weeks of therapy followed by one, two, three or four weeks of rest, or four weeks of therapy followed by one, two, three or four weeks of rest, or five weeks of therapy followed by one, two, three, four or five weeks of rest, or once every two weeks, once every three weeks, or once a month. The treatment can be extended for 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. The dosage can be up to 24 mg / kg. Typical dosage examples of administration may include 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg , 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg and 18 mg / kg. Preferred dosages are 4, 6, 8, 9, 10, 12, 14, 16 or 18 mg / kg. One of ordinary skill in the art will understand that various factors such as age, general health, specific organ function or weight, and the effect of prior therapy on certain organ systems (e.g., bone marrow) can be considered in determining the optimal dosage of the immunoconjugate, and that the dosage and / or frequency of administration can be increased or decreased during the course of therapy. The dosage can be repeated as needed, with tumor shrinkage observed after as little as 4 to 8 doses. The optimized dosages and dosing regimens disclosed herein demonstrate unexpected superior efficacy and reduced human toxicity that could not have been predicted from animal model studies. Surprisingly, the increased efficacy allows the treatment of tumors that have previously been found resistant to one or more standard anti-cancer therapies, including the parent compound CPT-11, from which SN-38 is generated in vivo.

[018] The methods of this invention may include the use of CT and / or PET / CT or MRI to measure tumor response at regular intervals. Levels of tumor markers in the blood such as CEA (fetal cancer antigen), CA19-9, AFP, CA 15.3, or PSA can also be monitored. Dosages and / or administration regimens can be adjusted as needed, in accordance with imaging results and / or blood marker levels.

[019] An unexpected result of the use of the compositions and methods disclosed herein is the unexpected tolerance of high doses of the antibody-drug conjugate, even with repeated infusions, with only relatively low levels of observed toxic effects in the form of nausea and vomiting or controlled neutropenia. Another unexpected result is the lack of accumulation of the antibody-drug conjugate, in contrast to other products in which SN-38 is conjugated to albumin, PEG or other carriers. The lack of accumulation is associated with improved tolerance and no serious toxicity even after repeated or increased dosing. These unexpected results allow the dosage and delivery schedule to be optimized with unexpectedly high efficacy and low toxicity. The claimed methods provide a reduction in the size of solid tumors in individuals with previously refractory cancers by up to 15% or more, preferably 20% or more, preferably 30% or more, more preferably 40% or more (as measured by the largest diameter). One of ordinary skill in the art will understand that tumor size can be measured in various ways, such as total tumor volume, maximum tumor size in any dimension, or a combination of dimensions in multiple dimensions. This can be done using standard X-ray procedures such as computed tomography, ultrasonography, and / or positron emission tomography. Means of measuring size are less important than observing the tendency for tumor size to decrease with immunoconjugate treatment, preferably leading to tumor disappearance.

[020] Although the immunoconjugate can be administered as a periodic bolus injection, in alternative embodiments, the immunoconjugate can be administered by continuous infusion of antibody-drug conjugates. To increase the Cmax and prolong the PK (pharmacokinetics) of the immunoconjugate in the blood, continuous infusion can be performed, for example, using an indwelling catheter. Such devices are known in the art, such as Hickman®, Broviac® or Port-A-Cath® catheters (see, for example, Skolnik et al., Ther Drug Monit 32: 741-48, 2010), and any such known indwelling catheter. A variety of continuous infusion pumps are also known to those skilled in the art, and any such known infusion pump may be used. The dose range for continuous infusion can be from 0.1 to 3.0 mg / kg per day. More preferably, these immunoconjugates can be administered by intravenous infusion over relatively short periods of time ranging from 2 to 5 hours, more preferably 2-3 hours.

[021] In particularly preferred embodiments, immunoconjugates and dosing regimens may be effective in patients refractory to standard therapies. For example, the anti-Trop-2 hRS7-SN-38 immunoconjugate can be administered to a patient who has not responded to prior therapy with irinotecan, the SN-38 parent agent. Surprisingly, an irinotecan-resistant patient may show a partial or even complete response to hRS7-SN-38. The ability of an immunoconjugate to specifically target tumor tissue can overcome tumor resistance through improved targeting and increased delivery of a therapeutic agent. CAP may also be effective for treating cancers that are resistant to other therapeutic agents, such as platinum-based anticancer agents. A particular preferred subject may be metastatic colon cancer patient, triple negative breast cancer patient, HER +, ER +, progesterone + breast cancer patient, metastatic non-small cell lung cancer (NSCLC) patient, metastatic pancreatic cancer patient, patient with metastatic renal cell carcinoma, patient with metastatic stomach cancer, patient with metastatic prostate cancer, patient with metastatic urothelial cancer, or patient with metastatic small cell lung cancer.

[022] In certain preferred embodiments, an antibody or immunoconjugate, such as sacituzumab govitecan, can be used in combination therapy with at least one microtubule inhibitor. A number of microtubule inhibitors are known in the art such as vinca alkaloids (eg vincristine, vinblastine), taxanes (eg paclitaxel), maytansinoids (eg mertansine) and auristatins. Other well-known microtubule inhibitors include demecolcine, nocodazole, epothilone, docetaxel, discodermolide, colchicine, combrestatin, podophyllotoxin, CI-980, phenylagistines, steganacins, curacins, 2-methoxyestradiol, E7010, dolonastinaminoxybenzenezene, rythroxystradiol, E7010, dolone aminaminoxybenzene, , tasidotin, halichondrins, hemiasterlins, cryptophycin 52, MMAE, and eribulin mesylate (see, for example, Dumontet & Jordan, 2010, Nat Rev Drug Discov 9: 790-803). Any such known microtubule inhibitor can be used in combination with an antibody or antibody-drug conjugate (ADC). Preferably, the microtubule inhibitor is a microtubule inhibitor that exhibits a synergistic effect when used in combination with an antibody or CAP. One effective example is an antibody conjugated to SN-38, such as sacituzumab govitecan or labetuzumab govitecan (targeting CEACAM5), expressed by many solid cancers. Most preferably, the microtubule inhibitor is paclitaxel or eribulin mesylate.

[023] In other preferred embodiments, the antibody or CAP can be used in combination therapy with at least one PARP inhibitor. A number of PARP inhibitors are known to those skilled in the art, such as olaparib, thalazoparib (BMN-673), rukaparib, veliparib, niraparib, iniparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699, BSI-201, CEP-8983 and 3 β-aminobenzamide (see, for example, Rouleau et al., 2010, Nat Rev Cancer 10: 293-301, Bao et al., 2015, Oncotarget [Epub ahead of print), September 22, 2015 ]). Any such known PARP inhibitor can be used in combination with an antibody or CAP, such as, for example, an SN-38 antibody conjugate. Preferably, the PARP inhibitor is an inhibitor that exhibits a synergistic effect when used in combination with an antibody or CAP. This has been confirmed using an antibody conjugated to SN-38 such as sacituzumab govitecan. Most preferably, the PARP inhibitor is olaparib or rucaparib.

[024] In other embodiments, the antibody or immunoconjugate can be used in combination with a Bruton's kinase inhibitor or PI3K inhibitor. Typical Bruton's kinase inhibitors include, without limitation, ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059, GDC-0834, LFM-A13, or RN486. Typical PI3K inhibitors include, but are not limited to, idelalisib, wortmannin, demethoxyviridine, perifosin, PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 or LY294002. Any Bruton's kinase or PI3K inhibitors known in the art can be used in the claimed combination therapy.

BRIEF DESCRIPTION OF THE GRAPHIC MATERIALS

[025] FIG. 1. Waterfall plot of the best responses in 6 patients with urothelial carcinoma treated with sacuzitumab govitecan. Clinical trials using IMMU-132 were performed as described in Example 1 below. Abbreviations used: PD is a progressive disease; CHO (PR) is a partial response; PC (Pt) represents a patient; RECIST is a criterion for evaluating the response of solid tumors; SD (SD) is a stable disease.

[026] FIG. 2A. Evaluation of target lesions on computed tomography (CT) scans using the criteria for evaluating the response of solid tumors, version 1.1, in patient (Pt) 6 before and after treatment with savituzumab govitecan. Patient 6 initially had 4 target lesions (2 in the liver, 1 in the sigmoid colon, 1 in the pelvic peritoneum) with the sum of the largest diameters of 138 mm. Additional off-target lesions were present in the liver and pelvic lymph node. Treatment was started at a dose level of 8 mg / kg. White arrows mark target lesions 1-3 in axial slices obtained at the beginning of the study. Axial slices of the same area 6 months after 9 cycles of treatment with sacituzumab govitecan showed a decrease in the total diameter of target lesions to 86 mm (minus 38%) and stable disease in non-target lesions.

[027] FIG. 2B. Expression of Trop-2 (TACSTD2) measured in three patients with urothelial carcinoma treated with sacuzitumab govitecan (patients 3, 4 and 6). Control slides were incubated with normal goat IgG. Corresponding sections incubated with goat anti-Trop-2 antibody showed strong expression of Trop-2. An enlarged insert with Trop-2 expressing cells (patient 6) demonstrated the localization of Trop-2 on cell membranes. The scale bars are 0.1 mm.

[028] FIG. 3. Phase I / II data of IMMU-132 for the best response according to RECIST criteria.

[029] FIG. 4. Responses in 52 TNBC (triple negative breast cancer) patients treated with 10 mg / kg IMMU-132 after multiple prior failures.

[030] FIG. 5. Progression-free survival in TNBC patients treated with 10 mg / kg IMMU-132.

[031] FIG. 6. Best response in 29 evaluated patients with NSCLC (non-small cell lung cancer) treated with 8 to 10 mg / kg IMMU-132.

[032] FIG. 7. Time to progression in patients with NSCLC treated with 8-10 mg / kg IMMU-132.

[033] FIG. 8. Progression-free survival in NSCLC patients treated with 8 or 10 mg / kg IMMU-132.

[034] FIG. 9A. Tumor growth inhibition by the combination of IMMU-132 and olaparib in TNBC: BRCA1 / 2- (protein of early development of breast cancer 1 or 2) and PTEN (homologue of phosphatase and tensin, deleted in chromosome 10) - defective tumors. Tumor-bearing mice (tumor volume (TV) about 0.3 cm ³ ) received olaparib (1 mg; about 50 mg / kg, intraperitoneally (ip) according to the MF regimen (drug administration from Monday to Friday); red arrows) or IMMU- 132 (intravenous (iv), weekly, black arrows). A non-tumor targeting anti-CD20 SN-38 CAP was used as a control. HCC1806 is a BRCA1 / 2-defective TNBC tumor line. Olaparib alone did not have a significant antitumor effect. IMMU-132 alone significantly inhibited tumor growth compared to all control groups (P <0.0106, AUC (area under curve)). IMMU-132 plus olaparib further improved antitumor responses compared to all groups (P <0.0019; AUC). Mice in the combination therapy group have yet to reach median survival (> 80.5 days), which is more than 2 and 4 times higher than those for IMMU-132 or olaparib monotherapy, respectively (P <0.0083) ...

[035] FIG. 9B. Tumor growth inhibition by the combination of IMMU-132 and olaparib in TNBC: BRCA1 / 2- (protein of early development of breast cancer 1 or 2) and PTEN (homologue of phosphatase and tensin, deleted in chromosome 10) - defective tumors. Tumor-bearing mice (tumor volume is about 0.3 cm ³⁾ was obtained olaparib (1 mg, about 50 mg / kg, i.p., on MF scheme (drug administration Monday through Friday); bright arrows) or IMMU-132 (intravenously weekly , dark arrows). A non-tumor targeting anti-CD20 SN-38 CAP was used as a control. In BRCA1 / 2 wild-type (wt), PTEN-defective MDA-MB-468 tumors, IMMU-132 alone produced significant antitumor effects compared to all control groups (P <0.0098; AUC). However, the combination of IMMU-132 plus olaparib inhibited tumor growth significantly better than either IMMU-132 or olaparib alone (P = 0.004; AUC). This leads to a significant increase in survival compared to all other groups (P less than 0.045).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[036] In the following description, a number of terms are used, and the following definitions are intended to facilitate an understanding of the subject matter of the claimed invention. Terms that are not expressly defined in this document are used in accordance with their simple and common meanings.

[037] Unless otherwise stated, terms in the singular (in the English text with articles a or an) mean “one or more”.

[038] The term is roughly used herein to mean plus or minus ten percent (10%) of a value. For example, “about 100” refers to any number between 90 and 110.

[039] An antibody, as used herein, refers to a full-length (i.e., naturally occurring or resulting from normal recombination processes of immunoglobulin gene fragments) immunoglobulin molecule (e.g., IgG antibody) or antigen-binding portion of an immunoglobulin molecule, such as a fragment antibodies. An antibody or antibody fragment can be conjugated or otherwise derivatized within the scope of the claimed invention. Such antibodies include, but are not limited to, IgG1, IgG2, IgG3, IgG4 (and IgG4 subforms), and IgA isotypes. As used below, the abbreviation "MAT" (MAb) can be used interchangeably with respect to an antibody, an antibody fragment, a monoclonal antibody, or a multispecific antibody.

[040] An antibody fragment is an antibody portion such as F (ab ') ₂ , F (ab) ₂ , Fab', Fab, Fv, scFv (single chain Fv), single domain antibodies (DAB or VHH) and the like. including the IgG4 "semi-molecules" mentioned above (van der Neut Kolfschoten et al. (Science 2007; 317 (14 Sept): 1554-1557). Regardless of the structure, the antibody fragment used binds to the same antigen recognized by the intact antibody. The term "antibody fragment" also includes synthetic or genetically engineered proteins that act as an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of variable regions, such as "Fv" fragments consisting of from the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules in which the light and heavy variable regions are linked by a peptide linker ("scFv proteins") Fragments can be constructed in various ways to obtaining multivalent and / or multispecific binding forms.

[041] A naked antibody is typically a whole antibody that is not conjugated to a therapeutic agent. Naked antibody can exhibit therapeutic and / or cytotoxic effects, for example, through Fc-dependent functions such as complement fixation (CDC, complement-dependent cytotoxicity) and ADCC (antibody-dependent cellular cytotoxicity). However, other mechanisms such as apoptosis, antiangiogenesis, antimetastatic activity, antiadhesive activity, inhibition of heterotypic or homotypic adhesion, and interference with signaling pathways can also provide a therapeutic effect. Naked antibodies include polyclonal and monoclonal antibodies, natural or recombinant antibodies such as chimeric, humanized or human antibodies and fragments thereof. In some cases, "naked" antibody can also refer to a "naked" fragment of an antibody. As used herein, "naked" is synonymous with "unconjugated" and means unbound or unconjugated to a therapeutic agent.

[042] A chimeric antibody is a recombinant protein that contains the variable domains of both heavy and light chains of an antibody, including the complementarity determining region (CDR) of an antibody derived from one species, preferably a rodent antibody, more preferably a murine antibody, while while the constant domains of an antibody molecule are derived from the constant domains of a human antibody. For veterinary use, the chimeric antibody constant domains can be derived from the constant domains of other species, such as primates, felines, or canines.

[043] A humanized antibody is a recombinant protein in which the CDR is from an antibody of the same species; for example, murine antibodies are transferred from the heavy and light variable chains of a murine antibody to the human heavy and light variable domains (framework regions). The constant domains of an antibody molecule are derived from the constant domains of a human antibody. In some cases, certain residues of the framework region of a humanized antibody, especially those adjacent to or close to the CDR sequences, can be modified, for example, replaced by corresponding residues from a mouse, rodent, apes, or other antibody.

[044] A human antibody is an antibody obtained, for example, from transgenic mice that have been "modified" to produce human antibodies in response to antigenic stimulation. In this method, elements of human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines that contain targeted breaks of endogenous heavy chain and light chain loci. Transgenic mice can synthesize human antibodies specific for various antigens, and mice can be used to generate hybridomas secreting human antibodies. 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). A fully human antibody can also be constructed by genetic or chromosomal transfection techniques, as well as phage display technology, which are all known to those of skill in the art. See, for example, McCafferty et al., Nature 348: 552-553 (1990), which describes in vitro production of human antibodies and fragments thereof, from immunoglobulin variable domain gene repertoires from non-immunized donors. In this method, the genes of the variable domain of a human antibody are cloned in frame into the gene of a major or minor coat protein of a filamentous bacteriophage and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particle contains a copy of the single-stranded DNA of the phage genome, selection based on the functional properties of the antibody also leads to the selection of the gene encoding the antibody exhibiting these properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, which are reviewed, for example, in Johnson and Chiswell, Current Opinion in Structural Biology 3: 5564-571 (1993). Human antibodies can also be generated in vitro by activated B cells. See US Pat. Nos. 5,567,610 and 5,229,275, the Examples sections of each of which are incorporated herein by reference.

[045] A therapeutic agent is an atom, molecule, or compound that is useful in treating a disease. Examples of therapeutic agents include, but are not limited to, antibodies, antibody fragments, immunoconjugates, drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases (including DNases and RNases), hormones, immunomodulators, chelating agents, boron compounds, photoactive agents or dyes, radionuclides, oligonucleotides, interference RNA, siRNA (small interfering RNA), RNAi (RNA interference), anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, or peptides that bind their combinations.

[046] An immunoconjugate is an antibody, an antigen-binding antibody fragment, an antibody complex, or an antibody fusion protein that is conjugated to a therapeutic agent. Conjugation can be covalent or non-covalent. Preferably, the conjugation is covalent. A particular form of immunoconjugate in which an antibody component is conjugated to a drug is referred to herein as an antibody drug conjugate (ADC).

[047] As used herein, the term antibody fusion protein means a recombinantly produced antigen binding molecule in which one or more natural antibodies, single chain antibodies or antibody fragments are linked to another fragment, such as a protein or peptide, toxin, cytokine, hormone etc. In certain preferred embodiments, the fusion protein can comprise two or more of the same or different antibodies, antibody fragments, or single chain antibodies fused together, which can bind to the same epitope, different epitopes on the same antigen, or different antigens.

[048] An immunomodulator is a therapeutic agent that, when present, alters, suppresses, or stimulates the body's immune system. Typically, the immunomodulator used stimulates the proliferation or activation of immune cells in the immune response cascade, such as macrophages, dendritic cells, B cells and / or T cells. However, in some cases, an immunomodulator can suppress the proliferation or activation of immune cells. An example of an immunomodulator described herein is a cytokine, which is a soluble small protein of approximately 5-20 kDa, released by a single population of cells (e.g., primed T lymphocytes) upon contact with specific antigens, and which acts as an intercellular mediator between cells ... One of ordinary skill in the art will understand that examples of cytokines include lymphokines, monokines, interleukins, and several related signaling molecules such as tumor necrosis factor (TNF) and interferons. Chemokines are a subset of cytokines. Some interleukins and interferons are examples of cytokines that stimulate the proliferation of T cells or other immune cells. Typical examples of interferons include interferon-α, interferon-β, interferon-γ, and interferon-λ.

[049] CPT is an abbreviation for camptothecin, and when used in this application, CPT refers to camptothecin itself or a camptothecin analog or derivative such as SN-38.

Anti-Trop2 antibodies

[050] Preferably, the CAP of this invention comprise at least one antibody or fragment thereof that binds to Trop-2. In a particularly preferred embodiment, the anti-Trop-2 antibody can be a humanized RS7 antibody (see, for example, U.S. Patent No. 7,238,785, incorporated herein by reference in its entirety) containing the CDR sequences of the light chain CDR1 (KASQDVSIAVA, SEQ ID NO: 1); CDR2 (SASYRYT, SEQ ID NO: 2); and CDR3 (QQHYITPLT, SEQ ID NO: 3) and CDR1 heavy chain CDR sequences (NYGMN, SEQ ID NO: 4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO: 5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO: 6).

[051] The RS7 antibody was a murine IgG ₁ against a crude membrane preparation of human primary squamous cell carcinoma of the lung (Stein et al., Cancer Res. 50: 1330, 1990). The RS7 antibody recognizes a 46-48 kDa glycoprotein characterized as cluster 13 (Stein et al., Int. J. Cancer Supp. 8: 98-102, 1994). The antigen has been designated EGP-1 (epithelial glycoprotein-1), but is also called Trop-2.

[052] Trop-2 is a type I transmembrane protein and has been cloned from both human (Fornaro et al., Int J Cancer 1995; 62: 610-8) and mouse cells (Sewedy et al., Int J Cancer 1998; 75: 324-30). It has been shown that, in addition to its role as a tumor-associated calcium signaling transporter (Ripani et al., Int J Cancer 1998; 76: 671-6), the expression of human Trop-2 is required for tumorigenesis and invasiveness of colon cancer cells, which can effectively reduce with a polyclonal antibody against the extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008; 7: 280-5).

[053] The growing interest in Trop-2 as a therapeutic target in solid cancers (Cubas et al., Biochim Biophys Acta 2009; 1796: 309-14) is further evidenced by reports documenting the clinical relevance of overexpressed Trop- 2 for breast carcinoma (Huang et al., Clin Cancer Res 2005; 11: 4357-64), colorectal cancer (Ohmachi et al., Clin Cancer Res 2006; 12: 3057-63; Fang et al., Int J Colorectal Dis 2009; 24: 875-84) and oral squamous cell carcinoma (Fong et al., Modern Pathol 2008; 21: 186-91). Of particular note is the recent evidence that basal prostate cells expressing high levels of Trop-2 are more likely to be enriched for in vitro and in vivo stem-like activity (Goldstein et al., Proc. Natl Acad Sci USA 2008; 105: 20882-7).

[054] Studies using flow cytometry and immunohistochemical staining have shown that the RS7 mAb detects antigen on various types of tumors, with limited binding to normal human tissue (Stein et al., 1990). Trop-2 is expressed primarily by carcinomas such as lung, stomach, bladder, breast, ovary, uterus, and prostate cancers. Localization and therapy studies using radioactively labeled murine MAb RS7 in animal models have demonstrated tumor targeting and therapeutic efficacy (Stein et al., 1990; Stein et al., 1991). Strong RS7 staining has been demonstrated in tumors of the lung, breast, bladder, ovary, uterus, stomach and prostate (Stein et al., Int. J. Cancer 55: 938, 1993). Lung cancer cases included both squamous cell carcinoma and adenocarcinoma (Stein et al., Int. J. Cancer 55: 938, 1993). Both types of cells were highly stained, indicating that the RS7 antibody does not distinguish between histological classes of non-small cell lung carcinoma.

[055] MAT RS7 is rapidly internalized by target cells (Stein et al., 1993). The rate constant of MAB internalization RS7 has a value intermediate between the rate constants of internalization of two other rapidly internalizing MABs, which have been demonstrated to be useful for the preparation of immunoconjugates (ibid.). It is well documented that the internalization of immunoconjugates is required for antitumor activity (Pastan et al., Cell 47: 641, 1986). The internalization of drug immunoconjugates has been described as an important factor in antitumor efficacy (Yang et al., Proc. Nat'l Acad. Sci. USA 85: 1189, 1988). Thus, the RS7 antibody exhibits several properties that are important for therapeutic use.

[056] Although the hRS7 antibody is preferred, other anti-Trop-2 antibodies are known and / or publicly available and, in alternative embodiments, can be used in the CAP of this invention. Although humanized or human antibodies are preferred for reducing immunogenicity, in alternative embodiments, a chimeric antibody can be used. As described below, methods for humanizing antibodies are well known in the art and can be used to convert a commercially available murine or chimeric antibody into a humanized form.

[057] Anti-Trop2 antibodies are commercially available from a variety of sources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417 (LifeSpan BioSciences, Inc., Seattle, WA); 10428-MM01, 10428-MM02, 10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54 (eBioscience, San Diego, CA); sc-376181, sc-376746, Santa Cruz Biotechnology (Santa Cruz, CA); MM0588-49D6, (Novus Biologicals, Littleton, CO); ab79976, and ab89928 (ABCAM®, Cambridge, MA).

[058] Other anti-Trop-2 antibodies have been disclosed in the patent literature. For example, US Publication No. 2013/0089872 discloses anti-Trop-2 antibodies K5-70 (FERM Accession No. BP-11251), K5-107 (FERM Accession No. BP-11252), K5-116-2-1 (FERM Accession No. BP-11253), T6-16 (FERM Accession No. BP-11346), and T5-86 (FERM Accession No. BP-11254) deposited with the International Patent Organism Depositary (Tsukuba, Japan). Patent No. 5840854 discloses an anti-Trop-2 monoclonal antibody BR110 (ATCC No. HB11698). US patent No. 7420040 discloses an anti-Trop-2 antibody produced by the hybridoma cell line AR47A6.4.2 deposited with the International Depository Authority of Canada (IDAC, Winnipeg, Canada) under Accession No. 141205-05. US patent No. 7420041 discloses an anti-Trop-2 antibody produced by the hybridoma cell line AR52A301.5 deposited with IDAC under Accession No. 141205-03. US Publication No. 2013/0122020 discloses anti-Trop-2 antibodies 3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representative antibody have been deposited with the American Type Culture Collection (ATCC), Accession Nos. PTA-12871 and PTA-12872. Immunoconjugate PF 06263507 containing an anti-5T4 (anti-Trop-2) antibody linked to a tubulin inhibitor monomethyluristatin F (MMAF) is available from Pfizer, Inc. (Groton, CT) (see, for example, Sapra et al., 2013, Mol Cancer Ther 12: 38-47). US Patent No. 8,715,662 discloses anti-Trop-2 antibodies produced by hybridomas deposited with AID-ICLC (International Depository Authority - Interlab Cell Lines Collection) (Genoa, Italy) with deposit numbers PD 08019, PD 08020 and PD 08021. Publication of a patent application US # 20120237518 discloses anti-Trop-2 antibodies 77220, KM4097 and KM4590. US patent No. 8309094 (Wyeth) discloses antibodies A1 and A3, identified by sequence listing. The Examples sections of each of the patents or patent applications referred to above in this paragraph are incorporated herein by reference. Non-patent publication Lipinski et al. (1981, Proc Natl. Acad Sci USA, 78: 5147-50) discloses anti-Trop-2 antibodies 162-25.3 and 162-46.2.

[059] Numerous antibodies against Trop-2 are known in the art and / or are generally available. As described below, methods for producing antibodies against known antigens have been routine in the art. The sequence of the human Trop-2 protein was also known in the art (see, for example, GenBank Accession No. CAA54801.1). Methods for producing humanized, human or chimeric antibodies have also been known. One of ordinary skill in the art, having read this disclosure in light of general knowledge in the art, could create and use a genus of anti-Trop-2 antibodies in the CAP of this invention.

Camptothecin conjugates

[060] Non-limiting methods and compositions for preparing immunoconjugates comprising a camptothecin therapeutic agent attached to an antibody or antigen binding fragment of an antibody are described below. In preferred embodiments, drug solubility is increased by placing a sized polyethylene glycol (PEG) moiety (i.e., PEG containing a specified number of monomer units) between the drug and the antibody, wherein said PEG is a low molecular weight PEG, preferably containing 1-30 monomer units, more preferably containing 1-12 monomer units, most preferably containing 6-8 monomer units.

[061] Preferably, the first linker is attached to the drug at one end and may terminate with an acetylene or azide group at the other end. This first linker may contain a sized PEG moiety with an azide or acetylene group at one end and another reactive group such as a carboxylic acid or hydroxyl group at the other end. The specified bifunctional PEG of a certain size can be attached to the amino group of the amino alcohol, and the hydroxyl group of the latter can be attached to the hydroxyl group of the drug in the form of carbonate. Alternatively, a non-azide (or acetylenic) moiety of said bifunctional PEG of a certain size is optionally attached to the N-terminus of an L-amino acid or polypeptide, and the C-terminus is attached to the amino group of the amino alcohol, and the hydroxy group of the latter is attached to the hydroxyl group of the drug in the form of a carbonate or carbamate, respectively ...

[062] A second linker containing an antibody-binding group and a reactive group complementary to the azide (or acetylenic) group of the first linker, namely acetylene (or azide), can react with the drug-first linker conjugate via an acetylene azide cycloaddition reaction to form the final bifunctional a drug product suitable for conjugation with disease-targeting antibodies. The antibody-binding group is preferably a thiol or a thiol-reactive group.

[063] Methods for selectively regenerating a 10-hydroxyl group in the presence of C-20 carbonate in drug-linker precursor formulations involving CPT analogs such as SN-38 are provided below. Other protecting groups for reactive hydroxyl groups in drugs can also be used, such as the phenolic hydroxyl in SN-38, for example, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, and are deprotected with tetrabutylammonium fluoride before binding the derivatized drug to the antibody binding The 10-hydroxyl group of the CPT analogs is alternatively protected as an ester or carbonate other than "BOC" (t-butyloxycarbonyl) so that the bifunctional CPT is conjugated to the antibody without first removing this protecting group. After the introduction of the bioconjugate, the protective group is easily removed at physiological pH.

[064] In a combination of acetylene azide called "click chemistry", the azide moiety can be on L2 and the acetylene moiety on L3. Alternatively, L2 may contain acetylene and L3 an azide. "Click chemistry" refers to the 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 are known and can be used. chemistry. Click chemistry takes place in an aqueous solution at close to neutral pH values and, therefore, is suitable for conjugation with a drug. The advantage of click chemistry is that it is chemoselective and complements other well known chemical conjugation methods such as the thiol-maleimide reaction.

[065] A typical preferred embodiment relates to a drug derivative and antibody conjugate of general formula (1) below.

MAT- [L2] - [L1] - [AA] _m - [A '] - Medicinal product (1)

where MAT is a disease targeting antibody; L2 is a cross-linking agent component containing an antibody-binding fragment and one or more acetylene (or azide) groups; L1 includes a PEG of a certain size with an azide (or acetylene) at one end complementary to the acetylene (or azide) moiety in L2, and a reactive group such as a carboxylic acid or hydroxyl group at the other end; AA stands for L-amino acid; m denotes an integer with the values 0, 1, 2, 3 or 4; and A 'is an additional spacer selected from the group of ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, or substituted or unsubstituted ethylenediamine. AA L-amino acids are selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and If the group A 'contains hydroxyl, it is bonded to the hydroxyl group or amino group of the drug in the form of a carbonate or carbamate, respectively.

[066] In a preferred embodiment of Formula 1, A 'is a substituted ethanolamine derived from an L-amino acid in which the carboxylic acid group of the amino acid is substituted with a hydroxymethyl moiety. A 'can 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.

[067] In an exemplary conjugate of a preferred embodiment of Formula 1, m is 0, A 'is L-valinol, and an exemplary drug is SN-38. In another example of formula 1, m is 1 and is represented by derivatized L-lysine, A 'is L-valinol, and an example of a drug is SN-38. In this embodiment, an amide bond is first formed between a carboxylic acid of an amino acid such as lysine and an amino group of valinol using orthogonal protecting groups for the amino group of lysine. The protecting group at the N-terminus of lysine is removed while keeping the protecting group intact in the lysine side chain, and the N-terminus is linked to a carboxyl group on a PEG of a certain size with an azide (or acetylene) at the other end. Then the hydroxyl group of valinol is attached to 20-chloroformate derivatives of 10-hydroxy-protected SN-38, and this intermediate is combined with the L2 component, which carries the antibody-binding fragment, as well as the complementary acetylene (or azide) group involved in the click chemistry of cycloaddition. Finally, removal of the protecting groups on both the lysine side chain and SN-38 gives the product of this example.

[068] Without wishing to be bound by any theory, the low molecular weight product of SN-38, namely valinol-SN-38-carbonate, formed after intracellular proteolysis, provides an additional pathway for the release of intact SN-38 through intramolecular cyclization involving the amino group of valinol and carbonyl carbonate.

[069] In another preferred embodiment, A 'of general formula 1 is A-OH, where A-OH is a collapsible moiety such as 4-aminobenzyl alcohol or substituted 4-aminobenzyl alcohol substituted with C ₁ -C ₁₀ an alkyl group at the benzyl position, and the latter, through its amino group, is attached to an L-amino acid or a polypeptide containing up to four L-amino acid fragments; wherein the N-terminus is attached to a cross-linking agent with a terminal antibody binding group.

[070] In another preferred embodiment, A-OH in A 'of general formula 1 is formed from substituted 4-aminobenzyl alcohol, and AA consists of one L-amino acid with m is 1 in general formula 1, and an example of a drug is SN -38. The individual amino acid AA can be selected 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. The R substituent on the 4-aminobenzyl alcohol moiety (embodiment A 'c A-OH) is hydrogen or an alkyl group selected from C1-C10 alkyl groups. An example of this formula, in which a single amino acid AA is L-lysine and R is H, and an example of a drug is SN-38, was designated MAT-CL2A-SN-38 (shown below). The structure differs from the MAT-CL2-SN-38 linker in that a single lysine residue is substituted for the Phe-Lys dipeptide present in the CL2 linker. The Phe-Lys dipeptide was designed as a cleavage site for cathepsin B by a lysosomal enzyme, which is considered important for the intracellular release of the bound drug. Surprisingly, despite the removal of the cathepsin cleavage site, immunoconjugates containing the CL2A linker appear to be more effective in vivo than those containing the CL2 linker.

[071] In a preferred embodiment, AA contains a polypeptide fragment, preferably a di-, tri- or tetrapeptide, which is cleaved by an intracellular peptidase. Examples are: Ala-Leu, Leu-Ala-Leu and Ala-Leu-Ala-Leu (SEQ ID NO: 7) (Trouet et al., 1982).

[072] In another preferred embodiment, the L1 component of the conjugate comprises a sized polyethylene glycol (PEG) spacer with 1-30 monomer repeating units. In a further preferred embodiment, the PEG is a defined size PEG with 1-12 monomer repeating units. Administration of PEG may involve the use of heterobifunctional PEG derivatives that are commercially available. Heterobifunctional PEG may contain an azide or acetylene group.

[073] In a preferred embodiment, L2 has a plurality of acetylene (or azide) groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5, and one antibody-binding fragment. In a representative example, the L2 component is attached to 2 acetylene groups resulting in the attachment of two azide-linked SN-38 molecules. Binding to MAT may involve succinimide.

[074] In preferred embodiments, when the bifunctional drug contains a thiol-reactive moiety as an antibody binding group, thiols in the antibody are generated at the lysine groups of the antibody using a thiolating reagent. Methods for introducing thiol groups into antibodies by modifying the lysine groups of the MAT are well known in the art (Wong, in Chemistry of protein conjugation and cross-linking, CRC Press, Inc., Boca Raton, FL (1991), pp 20-22). Alternatively, mild reduction of interchain disulfide bonds in an antibody (Willner et al., Bioconjugate Chem. 4: 521-527 (1993)) using reducing agents such as dithiothreitol (DTT) can generate 7 to 10 thiols in the antibody; its advantage is the inclusion of a plurality of drug fragments in the interchain region of the MAT, away from the antigen-binding region. In a more preferred embodiment, attachment of SN-38 to reduced disulfide sulfhydryl groups results in an antibody-SN-38 immunoconjugate with 6 covalently attached SN-38 fragments per antibody molecule. Other methods of preparing cysteine residues for attaching drugs or other therapeutic agents are known, such as the use of antibodies modified with cysteine (see US Pat. No. 7,521,541, the Examples section of which is incorporated herein by reference).

[075] In alternative preferred embodiments, the chemotherapeutic moiety is selected from the group consisting of doxorubicin (DOX), epirubicin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholinodoxorubicin (cyanomorpholino-DOX), 2-pyrrolinodoxorubicin (2-pyrrolinodoxorubicin) , CPT, 10-hydroxycamptothecin, SN-38, topotecan, lurtotecan, 9-aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin, ansamycins and epothilones. In a more preferred embodiment, the chemotherapeutic moiety is SN-38. Preferably, in the conjugates of preferred embodiments, the antibody binds to at least one chemotherapeutic moiety; preferably 1 to about 12 chemotherapeutic moieties; more preferably about 6 to about 12 chemotherapeutic moieties.

[076] In addition, in a preferred embodiment, the L2 linker component contains a thiol group that reacts with a thiol-reactive residue introduced into one or more amino groups of the lysine side chains of said antibody. In such cases, the antibody is pre-derivatized with a thiol-reactive group such as maleimide, vinyl sulfone, bromoacetamide, or iodoacetamide using procedures well described in the art.

[077] In the context of this work, a method has surprisingly been discovered by which it is possible to obtain drug-related CPT linkers, in which the CPT additionally contains a 10-hydroxyl group. This process includes, but is not limited to, protecting the 10-hydroxyl group as a t-butyloxycarbonyl (BOC) derivative, followed by the penultimate drug-linker conjugate intermediate. Usually, removal of the BOC group requires treatment with a strong acid such as trifluoroacetic acid (TFA). Under such conditions, the CPT 20-O-linker carbonate containing the protecting groups to be removed is also susceptible to degradation, resulting in the formation of unmodified CPT. In fact, the rationale for using a mildly removable methoxytrityl (MMT) protecting group for the lysine side chain of a linker molecule, as set forth in the art, was precisely to avoid this possibility (Walker et al., 2002). It has been found that selective removal of the phenolic protecting group BOC is possible by carrying out the reactions for short periods of time, optimally 3 to 5 minutes. Under these conditions, the predominant product was in which the BOC at the 10-hydroxyl position was removed, while the carbonate at the 20 position remained intact.

[078] An alternative approach involves protecting the 10-hydroxy position of the CPT analog with a group other than BOC so that the final product is ready for conjugation with antibodies without the need to remove the 10-OH protecting group. The 10-hydroxy protecting group, which converts 10-OH to phenolic carbonate or phenolic ester, is easily removed under physiological pH conditions or by esterases after in vivo administration of the conjugate. Faster removal of phenolic carbonate at position 10 compared to tertiary carbonate at position 20 of 10-hydroxycamptothecin under physiological conditions has been described by He et al. (He et al., Bioorganic & Medicinal Chemistry 12: 4003-4008 (2004)). The 10-hydroxy protecting group in SN-38 can be COR, where R can be substituted alkyl such as “N (CH ₃ ) ₂ - (CH ₂ ) _n -” where n is 1-10 and where the terminal amino group optionally in the form of a quaternary salt for increased water solubility or a simple alkyl moiety such as “CH ₃ - (CH ₂ ) _n -” where n is 0-10, or may be an alkoxy moiety such as “CH ₃ - (CH ₂ ) nO- "where n is 0-10, or" N (CH ₃ ) ₂ - (CH ₂ ) _n -O- "where n is 2-10, or" R ₁ O- (CH ₂ -CH ₂ -O) _n -CH ₂ -CH ₂ -O- ", where R ₁ is ethyl or methyl and n is an integer of 0-10. Such 10-hydroxy derivatives are readily prepared by treatment with chloroformate of the reagent of choice if the final derivative is to be a carbonate. Typically, a 10-hydroxy camptothecin such as SN-38 is treated with a molar equivalent of chloroformate in dimethylformamide using triethylamine as base. Under these conditions, the 20-OH position remains unchanged. The acid chloride of the selected reagent is used to form 10-O-esters.

[079] In a preferred method for preparing a conjugate of a drug derivative and an antibody of general formula 1, where the designations L2, L1, AA and AX have the meanings described in the previous sections, a bifunctional drug fragment is first obtained, [L2] - [L1] - [ AA] _m - [AX] -drug, followed by conjugation of a bifunctional fragment of the drug with an antibody (referred to herein as "MAT").

[080] In a preferred method for preparing a conjugate of a drug derivative and an antibody of general formula 1, where the designations L2, L1, AA and A-OH have the meanings described in the previous sections, a bifunctional drug moiety is prepared by first attaching A-OH to C- the end of AA via an amide bond, followed by coupling of the amine end of AA with the carboxylic acid group L1. If AA is absent (i.e., m is 0), A-OH is directly attached to L1 via an amide bond. The crosslinking agent, [L1] - [AA] _m - [A-OH], is attached to the hydroxyl or amino group of the drug, followed by attachment to the L1 moiety, using the reaction between the azide (or acetylenic) and acetylenic (or azide) groups in L1 and L2 by click chemistry methods.

[081] In one embodiment, the antibody is a monoclonal antibody (MAT). In other embodiments, the antibody can be a multivalent and / or multispecific mAb. The antibody can be a murine, chimeric, humanized or human monoclonal antibody, and said antibody can be intact, in the form of a fragment (Fab, Fab ', F (ab) ₂ , F (ab') ₂ ) or a sub-fragment (single chain constructs) , or belong to the isotype IgG1, IgG2a, IgG3, IgG4, IgA, or represent their submolecules.

[082] 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 hematopoietic tumor, carcinoma, sarcoma, melanoma or glial tumor cell. In a most preferred embodiment, the antibody fragment is anti-Trop-2 and anti-Trop-2-SN-38 CAP is used to treat any Trop-2 expressing cancer. In another preferred embodiment, the intracellularly cleavable fragment can be cleaved after its internalization into the cell upon binding of the MAT-drug conjugate to its receptor.

General antibody techniques

[083] Methods for producing monoclonal antibodies against virtually any target antigen are well known in the art. See, for example, Köhler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies can be generated by injecting mice with a composition containing the antigen, removing the spleen to obtain B lymphocytes, fusing B lymphocytes with myeloma cells to produce hybridomas, cloning hybridomas, selecting positive clones that produce antibodies to the antigen, culturing clones, producing antibodies to the antigen, and isolating antibodies from hybridoma cultures. One of ordinary skill in the art will understand that where antibodies are intended for administration to humans, the antibodies will bind to human antigens.

[084] MAT can be isolated and purified from hybridoma cultures using a variety of well-known methods. Such isolation techniques include Protein A or Protein G Sepharose affinity chromatography, size exclusion chromatography, and ion exchange chromatography. See, for example, Coligan on 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),” in: METHODS IN MOLECULAR BIOLOGY, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992).

[085] After the initial rise of antibodies to the immunogen, these antibodies can be sequenced and then produced by recombinant methods. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below.

[086] The skilled artisan will understand that any of a variety of antibodies known in the art can be used in the inventive methods and compositions. Useful antibodies are commercially available from a wide variety of known sources. For example, various antibody-secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA). A large number of antibodies against targets associated with various diseases, including, without limitation, tumor associated antigens, have been deposited with the ATCC and / or have published variable region sequences and are available for use in the claimed methods and compositions. See, for example, US Patent Nos. 7,312,318; 7282567; 7151164; 7074403; 7060802; 7056509; 7049060; 7045132; 7041803; 7041802; 7041293; 7038018; 7037498; 7012133; 7001598; 6998468; 6994976; 6994852; 6989241; 6974863; 6965018; 6964854; 6962981; 6962813; 6956107; 6951924; 6949244; 6946129; 6943020; 6939547; 6921645; 6921645; 6921533; 6919433; 6919078; 6916475; 6905681; 6899879; 6893625; 6887468; 6887466; 6884594; 6881405; 6878812; 6875580; 6872568; 6867006; 6864062; 6861511; 6861227; 6861226; 6838282; 6835549; 6835370; 6824780; 6824778; 6812206; 6793924; 6783758; 6770450; 6767711; 6764688; 6764681; 6764679; 6743898; 6733981; 6730307; 6720155; 6716966; 6709653; 6693176; 6692908; 6689607; 6689362; 6689355; 6682737; 6682736; 6682734; 6673344; 6653104; 6652852; 6635482; 6630144; 6610833; 6610294; 6605441; 6605279; 6596852; 6592868; 6576745; 6572856; 6566076; 6562618; 6545130; 6544749; 6534058; 6528625; 6528269; 6521227; 6518404; 6511665; 6491915; 6488930; 6482598; 6482408; 6479247; 6468531; 6468529; 6465173; 6461823; 6458356; 6455044; 6455040; 6451310; 6444206; 6441143; 6432404; 6432402; 6419928; 6413726; 6406694; 6403770; 6403091; 6395276; 6395274; 6387350; 6383759; 6383484; 6376654; 6372215; 6359126; 6355481; 6355444; 6355245; 6355244; 6346246; 6344198; 6340571; 6340459; 6331175; 6306393; 6254868; 6187287; 6183744; 6129914; 6120767; 6096289; 6077499; 5922302; 5874540; 5814440; 5798229; 5789554; 5776456; 5736119; 5716595; 5677136; 5587459; 5443953; 5525338, Sections of Examples of each of which are incorporated herein by reference. This listing is given by way of example only, and many other antibodies and their hybridomas are known in the art. One skilled in the art will understand that antibody sequences or hybridoma secreting antibodies against virtually any disease-associated antigen can be obtained by a simple search of the ATCC, NCBI (National Biotechnology Information Center) and / or USPTO (United States Patent and Trademark Office) databases. ) antibodies against the selected disease-associated target of interest. The antigen binding domains of cloned antibodies can be amplified, excised, ligated into an expression vector, transfected into an adapted host cell, and used to produce protein using standard techniques well known in the art. Isolated antibodies can be conjugated to therapeutic agents such as camptothecins using the techniques disclosed herein.

Chimeric and humanized antibodies

[087] A chimeric antibody is a recombinant protein in which the variable regions of a human antibody have been replaced with variable regions, for example, of a murine antibody, including the complementarity determining regions (CDRs) of a murine antibody. Chimeric antibodies exhibit reduced immunogenicity and increased stability when administered to a subject. Methods for constructing chimeric antibodies are well known in the art (eg, Leung et al., 1994, Hybridoma 13: 469).

[088] A chimeric monoclonal antibody can be humanized by transferring murine CDRs from the heavy and light variable chains of murine immunoglobulin into the corresponding variable domains of a human antibody. The mouse framework regions (FRs) in the chimeric monoclonal antibody are also replaced with human FR sequences. To maintain the stability and antigenic specificity of the humanized monoclonal (antibody), one or more human FR residues can be replaced with murine counterparts. Humanized monoclonal antibodies can be used to treat subjects therapeutically. Methods for producing humanized monoclonal antibodies are well known in the art (see, for example, 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).

[089] Other embodiments may relate to non-human primate antibodies. General methods for growing therapeutically useful antibodies in baboons are shown, for example, in Goldenberg et al., WO 91/11465 (1991) and Losman et al., Int. J. Cancer 46: 310 (1990). In another implementation, the antibody can be a human monoclonal antibody. Such antibodies can be obtained from transgenic mice that have been modified to produce specific human antibodies in response to antigenic stimulation, as described below.

Human antibodies

[090] Methods for producing fully human antibodies using either 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 Throughput Screen 8: 117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol. 3: 544-50; which are all incorporated herein by reference). It is expected that such fully human antibodies will exhibit even fewer side effects than chimeric or humanized antibodies, and will function in vivo as essentially endogenous human antibodies. In certain embodiments, the inventive methods and procedures can use human antibodies produced by such methods.

[091] In one alternative, a phage display technique can be used to generate human antibodies (eg, Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4: 126-40, incorporated herein by reference). Human antibodies can be generated in normal people or in people with manifestations of a certain disease condition, such as cancer (Dantas-Barbosa et al., 2005). An advantage of constructing human antibodies in a sick person is that the repertoire of circulating antibodies can be biased towards antibodies against disease-associated antigens.

[092] In one non-limiting example of this methodology, Dantas-Barbosa et al. (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Typically, total RNA was obtained from circulating blood lymphocytes (ibid.). Recombinant Fabs were cloned from µ-, γ- and κ-chain antibodies and inserted into a phage display library (ibid.). RNAs were converted to cDNA and used to generate Fab cDNA libraries using specific primers against immunoglobulin heavy and light chain sequences (Marks et al., 1991, J. Mol. Biol. 222: 581-97, incorporated herein by reference) ... Library construction was carried out in accordance with Andris-Widhopf et al. (2000, in: Phage Display Laboratory Manual, Barbas et al (eds), 1 ^{st edition, Cold Spring Harbor Laboratory Press} , Cold Spring Harbor, NY pp 9.1 to 9.22, incorporated herein by reference..). The final Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to create a phage display library. Such libraries can be screened using standard phage display techniques. One skilled in the art will understand that this technique is illustrative only, and any known method for producing and screening human antibodies or antibody fragments using phage display can be used.

[093] In another alternative, transgenic animals that have been genetically modified to produce human antibodies can be used to generate antibodies against virtually 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). A non-limiting example of such a system is XENOMOUSE® (eg, Green et al., 1999, J. Immunol. Methods 231: 11-23, incorporated herein by reference) from Abgenix (Fremont, CA). In XENOMOUSE® like 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.

[094] Transgenic mice were transformed with germline-configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Ig-kappa loci, including most of the variable region sequences, along with (along) accessory genes and regulatory sequences. The human variable region repertoire can be used to generate antibody-producing B cells that can be processed into hybridomas by known methods. XENOMOUSE® immunized with a target antigen will produce human antibodies through a normal immune response that can be harvested and / or produced by standard methods described above. Various strains of genetically modified mice are available, each producing a different class of antibodies. Transgenically produced human antibodies have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999). One skilled in the art will understand that the claimed compositions and methods are not limited to the use of the XENOMOUSE® system, but can use any transgenic animal that has been genetically modified to produce human antibodies.

Preparation of antibody fragments

[095] Some implementations of the claimed methods and / or compositions may relate to antibody fragments. Such antibody fragments can be obtained, for example, by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be prepared by enzymatic cleavage of antibodies with pepsin to generate a 5S fragment, designated F (ab ') ₂ . This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for sulfhydryl groups resulting from cleavage of disulfide bonds to obtain monovalent 3.5S Fab 'fragments. Alternatively, enzymatic digestion using pepsin yields two monovalent Fab fragments and an Fc fragment. Two Fab fragments can be covalently conjugated to form an F (ab) ₂ antibody fragment. Exemplary methods for making antibody fragments are disclosed in US Pat. No. 4,036,945; US patent No. 4331647; Nisonoff et al., 1960, Arch. Biochem. Biophys. 89: 230; Porter, 1959, Biochem. J. 73: 119; Edelman et al., 1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

[096] Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent fragments of light-heavy chains, further cleavage of fragments, or other enzymatic, chemical or genetic methods, can also be used, provided that the fragments bind to the antigen recognized by the intact antibody ... For example, Fv fragments contain an association of V _H and V _L chains. This association can be non-covalent, as described in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA 69: 2659. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked with chemical reagents such as glutaraldehyde. See Sandhu, 1992, Crit. Rev. Biotech. 12: 437.

[097] Preferably, the Fv fragments contain V _H and V _L chains connected by a peptide linker. Such single-stranded antigen binding proteins (scFvs) are obtained by constructing a structural gene containing DNA sequences encoding V _H and V _L domains connected by an oligonucleotide linker sequence. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide linking two V domains. Methods for preparing scFvs are well known in the art. See Whitlow et al., 1991, Methods: A Companion to Methods in Enzymology 2:97; Bird et al. 1988 Science 242: 423; U.S. Patent No. 4,946,778; Pack et al., 1993, Bio / Technology, 11: 1271, and Sandhu, 1992, Crit. Rev. Biotech. 12: 437.

[098] Another form of an antibody fragment is a single domain antibody (dAb), which is sometimes referred to as a single chain antibody. Methods for producing single domain antibodies are well known in the art (see, for example, 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: 24774-780). Other types of antibody fragments may contain one or more complementarity determining regions (CDRs). CDR peptides ("minimum recognition units") can be obtained by constructing genes encoding the CDRs of the antibody of interest. Such genes are obtained, for example, by using the polymerase chain reaction to synthesize the variable region from the RNA of antibody-producing cells. See Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2: 106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (Cambridge University Press); Birch et al., (Eds.), 1995, MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody variants

[099] In certain embodiments, antibody sequences, such as Fc regions of antibodies, can be varied to optimize the physiological characteristics of the conjugates, such as serum half-life. Methods for the replacement amino acid sequences in proteins are widely known in the art, such as site-directed mutagenesis (e.g., Sambrook et al., Molecular Cloning , A laboratory manual, 2 ^nd Ed, 1989). In preferred embodiments, changes may include the addition or deletion of one or more glycosylation sites in the Fc sequence (eg, US Pat. 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; US Patent No. 7,217,797; all of which are incorporated herein by reference).

Target antigens and typical antibodies

[0100] In a preferred embodiment, antibodies are used that recognize and / or bind to antigens that are expressed at high levels on target cells and that are expressed predominantly or exclusively on diseased cells as compared to normal tissues. More preferably, the antibodies are rapidly internalized upon binding. A typical rapidly internalizing antibody is LL1 (anti-CD74), with an internalization rate of approximately 8 × 10 ⁶ antibody molecules per cell per day (eg, Hansen et al., 1996, Biochem J. 320: 293-300). Thus, a "rapidly internalizing" antibody can be an antibody with an internalization rate of about 1 x 10 ⁶ to about 1 x 10 ⁷ antibody molecules per cell per day. Antibodies suitable for use in the claimed compositions and methods may include MABs with the properties indicated above. Typical antibodies used for therapy, for example, cancer, include, without limitation, LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab ( GA101, anti-CD20), lambrolizumab (against PD-1 receptor), nivolumab (against PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-Trop-2), PAM4 or KC4 (both against mucin ), MN-14 (against embryonic carcinoma antigen (CEA, also known as CD66e or CEACAM5)), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (against colon-specific antigen-p), Immu 31 (anti-alpha fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g. CC49), Tn, J591, or HuJ591 (anti-PSMA (prostate specific membrane antigen)) , AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX MAT), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab (anti -VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20); PAM4 (also clivatuzumab, anti-MUC5ac) and trastuzumab (anti-ErbB2). Such antibodies are known to those skilled in the art (for example, US Patent Nos. 5686072; 5874540; 6107090; 6183744; 6306393; 6653104; 6730300; 6899864; 6926893; 6962702; 7074403; 7230084; 7238785; 7238786; 7256004; 7282567; 7300; 7300; 7256004; 7282567; 7300 ; 7642239; and US Patent Application Publication Nos. 20050271671; 20060193865; 20060210475; 20070087001; Examples sections of each of which are incorporated herein by reference). Specific known useful antibodies include hPAM4 (US Pat. No. 7,282,567), hA20 (US Pat. No. 7151164), hA19 (US Pat. No. 7,109,304), hIMMU-31 (US Pat. No. 7,300,655), hLL1 (US Pat. No. 7,312,318), hLL2 (US patent No. 5789554), hMu-9 (US patent No. 7387772), hL243 (US patent No. 7612180), hMN-14 (US patent No. 6676924), hMN-15 (US patent No. 8287865), hR1 (US patent No. 9441043), hRS7 (US patent No. 7238785), hMN-3 (US patent No. 7541440), AB-PG1-XG1-026 (US patent application 11/983372, deposited as ATCC PTA-4405 and PTA-4406) and D2 / B (WO 2009/130575), where the text of each cited patent or application is incorporated herein by reference in relation to the Figures and Examples sections. In a particularly preferred embodiment, the antibody is hRS7.

[0101] Other suitable antigens that may be targeted using the described conjugates include carbonic anhydrase IX, B7, CCL19, CCL21, CSAp, HER-2 / neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g. C2B8, hA20, MAT 1F5), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4, alpha-fetoprotein ( AFP), VEGF (e.g. bevacizumab, fibronectin splice variant), ED-B fibronectin (e.g. L19), Trop-2, EGP-2 (e.g. 17-1A), EGF receptor (ErbB1) ( e.g. cetuximab), ErbB2, ErbB3, factor H, FHL-1, Flt-3, folate receptor, Ga 733, GRO-β, HMGB-1, hypoxia-inducible factor (HIF), HM1.24, HER-2 / neu, insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL -18R, IL-2, I L-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, HLA-DR antigen to which L243 binds, CD66 antigens, i.e., CD66a-d or a combination thereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration inhibiting factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor (PlGF), PSA (prostate specific antigen), PSMA, PAM4 antigen, PD-1, PD-L1, NCA-95, NCA-90, A3, A33, Ep-CAM, KS -1, Le (y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumor necrosis antigens, tumor angiogenesis antigens, TNF-α, TRAIL receptor (R1 and R2), Trop -2, VEGFR, RANTES, T101, as well as cancer stem cell antigens, complement factors C3, C3a, C3b, C5a, C5, and an oncogene product.

[0102] Comprehensive analysis of suitable target antigens (cluster designation, or CD) on hematopoietic malignant cells, as shown by flow cytometry, which can serve as a guide for the selection of suitable antibodies for immunotherapy with drug conjugates, provided by Craig and Foon, Blood, Web Preview January 15, 2008; DOL 10.1182 / blood-2007-11-120535.

[0103] The CD66 antigens are composed of five different glycoproteins with similar structures, CD66a-e, encoded by members of the embryonic cancer antigen (CEA) gene family BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66 antigens (eg, CEACAM6) are expressed mainly in granulocytes, normal epithelial cells of the digestive tract, and tumor cells of various tissues. Testicular cancer antigens such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007; 120 (11): 2411-7) and CD79a in myeloid leukemia (Kozlov et al. ., Cancer Genet. Cytogenet. 2005; 163 (1): 62-7) and also B-cell diseases and CD79b in non-Hodgkin's lymphoma (Poison et al., Blood 110 (2): 616-623). A number of the aforementioned antigens are disclosed in Provisional Application No. 60/426379, entitled Use of Multi-specific, Non-covalent Complexes for Targeted Delivery of Therapeutics, filed November 15, 2002. cells that are described as being more resistant to therapy in the malignant progenitor cell population (Hill and Perris, J. Natl. Cancer Inst. 2007; 99: 1435-40) have antigens that can target certain types of cancer, such as CD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos. Proc. 2006; 5: 155-79), 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 CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104 (24) 10158-63), pancreatic cancer (Li et al., Cancer Res. 2007; 67 (3): 1 030-7), and squamous cell carcinoma of the head and neck (Prince et al., Proc. Natl. Acad. Sci. USA 2007; 104 (3) 973-8). Another suitable target for breast cancer therapy is the LIV-1 antigen described by Taylor et al. (Biochem. J. 2003; 375: 51-9). The CD47 antigen is an additional useful target for cancer stem cells (see, for example, Naujokat et al., 2014, Immunotherapy 6: 290-308; Goto et al., 2014, Eur J Cancer 50: 1836-46; Unanue, 2013, Proc Natl Acad Sci USA 110: 10886-7).

[0104] Suitable targeting antibodies against, for example, CD38 and CD138 have been described for the treatment of multiple myeloma (Stevenson, Mol Med 2006; 12 (11-12): 345-346; 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).

[0105] Antibodies of checkpoint inhibitors have been used in cancer therapy. Immune checkpoints refer to inhibitory pathways in the immune system that are responsible for maintaining self-tolerance and modulating the degree of response of the immune system to minimize damage to peripheral tissues. However, tumor cells can also activate checkpoints of the immune system to reduce the effectiveness of the immune response against tumor tissues. Typical examples of checkpoint inhibitor antibodies against cytotoxic T-lymphocyte-4 antigen (CTLA4, also known as CD152), programmed cell death protein 1 (PD-1, also known as CD279) and programmed cell death ligand 1 (protein) 1 (PD -L1, also known as CD274), can be used in combination with one or more other agents to enhance the effectiveness of the immune response against cells, tissues, or disease pathogens. Typical examples of anti-PD-1 antibodies include lambrolizumab (MK-3475, Merck), nivolumab (BMS-936558, Bristol-Myers Squibb), AMP-224 (Merck), and pidilizumab (CT-011, CURETECH LTD.). Anti-PD-1 antibodies are commercially available, for example from Abcam® (ab137132), BIOLEGEND® (EH12.2H7, RMP1-14) and Affymetrix ebioscience (j105, j116, mih4). Typical examples of anti-PD-L1 antibodies include MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH), and BMS-936559 (Bristol-Myers Squibb). Anti-PD-L1 antibodies are also commercially available, for example from AFFYMETRIX EBIOSCIENCE (MIH1). Typical examples of anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer). Anti-PD1 antibodies are commercially available, for example from Abcam® (ab134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572, PA5-23967, PA5-26465, MA1-12205, MA1-35914). Ipilimumab recently received FDA approval for the treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11:89).

[0106] Macrophage migration inhibition factor (MIF) is an important regulator of innate and adaptive immunity and apoptosis. CD74 has been reported to be an endogenous MIF receptor (Leng et al., 2003, J Exp Med 197: 1467-76). The therapeutic effect of antagonistic anti-CD74 antibodies on MIF-mediated intracellular pathways may be useful in the treatment of a wide range of disease conditions, such as cancers of the bladder, prostate, breast, lung, colon, 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: e79-83); and numerous inflammatory diseases (Meyer-Siegler et al., 2009, Mediators Inflamm epub March 22, 2009; Takahashi et al., 2009, Respir Res 10:33; milatuzumab (hLL1) is a typical exemplary anti-CD74 antibody useful for therapeutic use in the treatment of MIF-mediated diseases.

[0107] Anti-TNF-α antibodies are known in the art and can be used to treat a variety of diseases. Known antibodies against TNF-α include the human antibody CDP571 (Ofei et al., 2011, Diabetes 45: 881-85); mouse antibodies MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, and M303 (Thermo Scientific, Rockford, IL); infliximab (Centocor, Malvern, PA); certolizumab pegol (UCB, Brussels, Belgium); and adalimumab (Abbott, Abbott Park, IL). These and many other known anti-TNF-α antibodies can be used in the claimed methods and compositions.

[0108] In another preferred embodiment, antibodies are used that are rapidly internalized and then re-expressed, processed, and presented on cell surfaces, allowing continuous uptake by the cell and formation of accumulations (accretion) of the circulating conjugate. An example of a most preferred antibody / antigen pair is LL1, anti-CD74 MAT (invariant chain, class II specific chaperone, Ii) (see, for example, U.S. Patent Nos. 6,653,104; 7,312,318; Examples sections of each of which are incorporated herein by reference ). The CD74 antigen is expressed at high levels on B-cell lymphomas (including multiple myeloma) and leukemias, some T-cell lymphomas, melanomas, colon, lung and kidney cancers, glioblastomas, and several other cancers (Ong et al., Immunology 98: 296-302 (1999)). For a review of the use of antibodies against CD74 in cancer, see Stein et al., Clin Cancer Res. 2007 Sep 15; 13 (18 Pt 2): 5556s-5563s, and is incorporated herein by reference.

[0109] Diseases that are preferably treated with antibodies against CD74 include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, melanoma, cancers of the lung, kidney, colon, glioblastoma multiforme, histiocytoma, myeloid leukemia, and multiple myeloma. Continuous expression of the CD74 antigen for short periods of time on the surface of target cells, followed by internalization of the antigen and re-expression of the antigen makes it possible for the targeting antibody LL1 to internalize along with any chemotherapeutic fragment it carries. This allows a high and therapeutic concentration of the LL1-chemotherapeutic drug conjugate to accumulate within such cells. Internalized LL1-chemotherapeutic drug conjugates cyclically pass through the lysosomes and endosomes and the chemotherapeutic moiety is released in active form in the target cells.

[0110] The above antibodies and other known antibodies against disease-associated antigens can be used as CPT conjugates, more preferably SN-38 conjugates, in the practice of the claimed methods and compositions. In a most preferred embodiment, the drug-conjugated antibody is an anti-Trop-2-SN-38 conjugate antibody (eg, hRS7-SN-38).

Bispecific and multispecific antibodies

[0111] Bispecific antibodies are useful in a number of biomedical applications. For example, a bispecific antibody with tumor cell surface antigen and T cell surface receptor binding sites can direct T cell lysis of certain tumor cells. Bispecific antibodies that recognize gliomas and the CD3 epitope on T cells have been used successfully in the treatment of human brain tumors (Nitta, et al. Lancet. 1990; 355: 368-371). The preferred bispecific antibody is an anti-CD3x anti-Trop-2 antibody. In alternative embodiments, an anti-CD3 antibody or fragment thereof can be linked to an antibody or fragment against an antigen associated with a B cell, e.g., anti-CD3 x anti-CD19, anti-CD3 x anti-CD20, anti-CD3 x anti-CD22, anti-CD3x anti-HLA-DR, or anti-CD3x anti-CD74. In certain embodiments, the methods and compositions for conjugating a therapeutic agent disclosed herein can be used with bispecific or multispecific antibodies as targeting fragments.

[0112] Numerous methods for producing bispecific or multispecific antibodies are known, as disclosed, for example, in US Pat. No. 7,405,320, the Examples section of which is incorporated herein by reference. Bispecific antibodies can be produced by the quadroma method, which involves the fusion of two different hybridomas, each producing a monoclonal antibody that recognizes different antigenic sites (Milstein and Cuello, Nature, 1983; 305: 537-540).

[0113] Another method for producing bispecific antibodies uses heterobifunctional crosslinking agents to chemically link two different monoclonal antibodies (Staerz, et al. Nature, 1985; 314: 628-631; Perez, et al. Nature, 1985; 316: 354-356) ... Bispecific antibodies can also be prepared by reducing each of the two parental monoclonal antibodies to their respective semimolecules, which are then mixed and allowed to re-oxidize to produce a hybrid structure (Staerz and Bevan. Proc Natl Acad Sci US A. 1986; 83: 1453-1457 ). Another alternative involves chemical crosslinking of two or three separately purified Fab 'fragments using appropriate linkers (see, for example, European patent application 0453082).

[0114] Other methods include increasing the efficiency of creating hybrid hybridomas by transferring genes of various selectable markers using retroviral-derived shuttle vectors into corresponding parental hybridomas, which are subsequently fused (DeMonte, et al. Proc Natl Acad Sci US A. 1990, 87: 2941 -2945); or transfection of a hybridoma cell line with expression plasmids containing genes for the heavy and light chains of another antibody.

[0115] Related V _H and V _L domains can be linked by a peptide linker of appropriate composition and length (usually more than 12 amino acid residues) to form a single chain Fv (scFv) with binding activity. Methods for preparing scFv are disclosed in US Pat. No. 4,946,778 and US Pat. No. 5,132,405, the Examples sections of each of which are incorporated herein by reference. Reducing the length of the peptide linker to less than 12 amino acid residues prevents pairing of the V _H and V _L domains in one chain and leads to pairing of the V _H and V _L domains with complementary domains of other chains, leading to the formation of functional multimers. The polypeptide chains of the V _H and V _L domains, which are connected by linkers ranging in size from 3 to 12 amino acid residues, form predominantly dimers (called diabodies). With linkers ranging in size from 0 to 2 amino acid residues, trimers (called tri-bodies) and tetramers (called tetrabodies) are preferred, but the precise oligomerization patterns seem to depend on the composition as well as the orientation of the V-domains (V _H -linker-V _L or V _L -linker-V _H ), in addition to the length of the linker.

[0116] These methods for producing multispecific or bispecific antibodies have various difficulties in terms of low yield, purification, low stability, or laboriousness of the method. More recently, a technique known as dock and lock (DNL®) has been used to produce combinations of virtually any desired antibody, antibody fragment, and other effector molecule (see, eg, US Pat. Nos. 7,521,056; 7,527,787; 7534866; 7550143; 7666400; 7858070; 7871622; 7906121; 7906118; 8163291; 7901680; 7981398; 8003111 and 8034352, sections of the Examples of each of which are incorporated by reference herein.) This method uses complementary protein binding domains called anchor domains (AD) and dimerization and docking domains (DDDs) that bind to each other and allow complex structures ranging from dimers, trimers, tetramers, quintamers and hexamers to be assembled to form stable complexes in high yields without the need for extensive purification. allows the collection of monospecific, bispecific or multispecific antibodies Any methods known in the art for obtaining bispecific or multispecific antibodies Ific antibodies can be used in the practice of the methods disclosed herein.

Antibody allotypes

[0117] The immunogenicity of therapeutic antibodies is associated with an increased risk of infusion reactions and a decrease in the duration of the therapeutic response (Baert et al., 2003, N Engl J Med 348: 602-08). The extent to which therapeutic antibodies induce an immune response in a host may in part be determined by the allotype of the antibody (Stickler et al., 2011, Genes and Immunity 12: 213-21). An antibody allotype is associated with amino acid sequence variations at specific positions in the antibody constant region sequences. Allotypes of IgG antibodies containing the heavy chain γ-type constant region are referred to as Gm allotypes (1976, J Immunol 117: 1056-59).

[0118] For common human IgG1 antibodies, the most common allotype is G1m1 (Stickler et al., 2011, Genes and Immunity 12: 213-21). However, the G1m3 allotype is also common in Caucasians (ibid.). G1m1 antibodies have been reported to contain allotype sequences that tend to induce an immune response when administered to non-G1m1 (nG1m1) recipients such as patients G1m3 (ibid.). Antibodies other than the G1m1 allotype are not immunogenic when given to patients with G1m1 (ibid.).

[0119] The human allotype G1m1 contains the amino acids aspartic acid at position 356 Kabat and leucine at position 358 Kabat in the CH3 sequence of the IgG1 heavy chain. The nG1m1 allotype contains the amino acids glutamic acid at position 356 Kabat and methionine at position 358 Kabat. Both the G1m1 and nG1m1 allotypes contain a glutamic acid residue at position 357 Kabat, and these allotypes are sometimes referred to as DEL and EEM allotypes. A non-limiting example of heavy chain constant region sequences for antibodies of allotypes G1m1 and nG1m1 is shown for typical antibodies rituximab (SEQ ID NO: 8) and veltuzumab (SEQ ID NO: 9).

Rituximab Heavy Chain Variable Region Sequence (SEQ ID NO: 8)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Veltuzumab Heavy Chain Variable Region (SEQ ID NO: 9)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0120] Jefferis and Lefranc (2009, mAbs 1: 1-7) review the sequence variations characteristic of IgG allotypes and their effect on immunogenicity. They reported that the G1m3 allotype is characterized by an arginine residue at position 214 Kabat, compared to a lysine residue at position 214 Kabat in allotype G1m17. The nG1m1,2 allotype was characterized by glutamic acid at position 356 according to Kabat, methionine at position 358 according to Kabat, and alanine at position 431 according to Kabat. Allotype G1m1,2 was characterized by aspartic acid at position 356 Kabat, leucine at position 358 Kabat and glycine at position 431 Kabat. In addition to heavy chain constant region sequence variants, Jefferis and Lefranc (2009) reported allotype variants in the kappa light chain constant region, with the Km1 allotype characterized by valine at position 153 Kabat and leucine at position 191 Kabat, allotype Km1,2 by alanine at position 153 Kabat and leucine at position 191 Kabat, and the Km3 allotype is characterized by alanine at position 153 Kabat and valine at position 191 Kabat.

[0121] With regard to therapeutic antibodies, veltuzumab and rituximab are, respectively, humanized and chimeric IgG1 antibodies against CD20, which are used to treat a wide range of hematological malignancies. Table 1 shows a comparison of the allotype sequences of rituximab and veltuzumab. As shown in Table 1, rituximab (G1m17.1) is a DEL-allotype of IgG1, with an additional sequence change at position 214 on Kabat (CH1 of the heavy chain) from lysine in rituximab to arginine in veltuzumab. Veltuzumab has been reported to be less immunogenic in humans than rituximab (see, for example, Morchhauser et al., 2009, J Clin Oncol 27: 3346-53; Goldenberg et al., 2009, Blood 113: 1062-70; Robak & Robak, 2011, BioDrugs 25: 13-25), and this effect has been attributed to the difference between humanized and chimeric antibodies. However, the differences between the EEM and DEL allotypes probably also explain the lower immunogenicity of veltuzumab.

Table 1. Comparison of the allotypes of rituximab and veltuzumab

Heavy chain position and associated allotypes Full allotype 214 (allotype) 356/358 (allotype) 431 (allotype) Rituximab G1m17.1 K 17 D / L 1 A - Veltuzumab G1m3 R 3 E / M - A -

[0122] To reduce the immunogenicity of therapeutic antibodies in individuals with the nG1m1 genotype, it is desirable to select an antibody allotype corresponding to the allotype G1m3, characterized by arginine at position 214 according to Kabat, and the null allotype nG1m1,2, characterized by glutamic acid at position 356 at position Kabat, methionine 358 Kabat and Alanine at 431 Kabat. Surprisingly, it was found that repeated subcutaneous administration of G1m3 antibodies over a long period of time did not result in a significant immune response. In alternative embodiments, the human IgG4 heavy chain comprises arginine at position 214 Kabat, glutamic acid at position 356 Kabat, methionine at position 359 Kabat, and alanine at position 431 Kabat, as well as allotype G1m3. Since immunogenicity appears to be related, at least in part, to residues at these positions, the use of the human IgG4 heavy chain constant region sequence for therapeutic antibodies is also a preferred embodiment. Combinations of IgG1 G1m3 antibodies with IgG4 antibodies can also be used for therapeutic administration.

Conjugation protocols

[0123] Antibodies or fragments thereof can be conjugated to one or more therapeutic or diagnostic agents. Therapeutic agents do not have to be the same, but can be different, for example, a drug and a radioisotope. For example, ¹³¹ I can be incorporated into the tyrosine of an antibody or fusion protein, and the drug is attached to the epsilon amino group of a lysine residue. Therapeutic and diagnostic agents can also be attached, for example, to reduced SH groups and / or carbohydrate side chains. Many methods are known in the art for preparing covalent or non-covalent conjugates of therapeutic or diagnostic agents with antibodies or fusion proteins, and any such known method can be used.

[0124] A therapeutic or diagnostic agent can be attached to the hinge region of the recovered antibody component by disulfide bond formation. Alternatively, such agents can be coupled using a heterobifunctional crosslinking agent such as N-succinyl-3- (2-pyridyldithio) propionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). General methods for such conjugation are well known to those skilled in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification of Antibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995). Alternatively, a therapeutic or diagnostic agent can be conjugated via a carbohydrate moiety in the Fc region of an antibody. The carbohydrate group can be used to increase the content of the same agent as that linked to the thiol group, or the carbohydrate group can be used to link another therapeutic or diagnostic agent.

[0125] Methods for conjugating peptides to antibody components via an antibody carbohydrate fragment are well known to those of skill in the art. See, for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al., Int. J. Cancer 46: 1101 (1990); and Shih et al., US Pat. No. 5,057,313, incorporated herein in its entirety by reference. The general method includes contacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine function. This reaction results in the formation of an initial Schiff base (imine) bond, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0126] The Fc region may be absent if the antibody used as an antibody component of the immunoconjugate is an antibody fragment. However, you can introduce a carbohydrate fragment into the variable region of the light chain of a full-length antibody or antibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., US patent No. 5443953 (1995), Leung et al., US patent No. 6254868, which are incorporated herein by reference in their entirety. The engineered carbohydrate moiety is used to attach a therapeutic or diagnostic agent.

[0127] The preferred conjugation protocol is based on the reaction thiol-maleimide, thiol-vinyl sulfone, thiol-bromoacetamide, or thiol-iodoacetamide, which readily proceeds at neutral or acidic pH. This eliminates the need for higher pH values for conjugation, such as would be required with active esters. Additional details of exemplary conjugation protocols are described below in the Examples section.

Therapeutic treatment

[0128] In another aspect, the invention relates to a method of treating a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate (ADC) as described herein. Diseases that can be treated with the PACs 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 lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia), using, for example, an anti-CD22 antibody such as mAb hLL2 (epratuzumab, see US patent No. 6183744), against another epitope CD22 (hRFB4), or antibodies against other antigens B -cells such as CD19, CD20, CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD74, CD80 or HLA-DR. Other diseases include, but are not limited to, adenocarcinomas of the endodermic epithelium of the digestive system, cancers such as breast cancer and non-small cell lung cancer, and other carcinomas, sarcomas, glial tumors, myeloid leukemias, etc. In particular, antibodies against an antigen, for example, an oncofetal antigen produced or associated with a malignant solid tumor or neoplasm of the hematopoietic system, for example, a tumor of the gastrointestinal tract, stomach, colon, esophagus, liver, lung, breast, pancreas, are advantageously used. liver, prostate, ovary, testicle, brain, bone, urothelium or lymphatic system, sarcoma, or melanoma. Such therapeutic agents can be administered one or more times, depending on the disease state and tolerance of the conjugate, and can also be used, optionally, in combination with other therapeutic methods such as surgery, external radiation, radioimmunotherapy, immunotherapy, chemotherapy, antisense therapy, therapy RNA interference, gene therapy, etc. Each combination will adapt to the type of tumor, stage, condition of the patient and previous therapy, as well as other factors considered by the treating physician.

[0129] As used herein, the term "subject" refers to any animal (ie, vertebrates and invertebrates), including, without limitation, mammals, including humans. The term is not intended to be limited to a specific age or gender. Thus, the term encompasses adults and newborns as well as fetuses, both male and female. The doses given in this document are intended for humans, but may be adjusted to suit the size of other mammals, as well as children, based on weight or size in square meters.

[0130] In a preferred embodiment, therapeutic conjugates containing an anti-Trop-2 antibody, such as MAT hRS7, can be used to treat carcinomas such as cancer of the esophagus, pancreas, lung, stomach, colon and rectum, bladder, breast gland, ovary, uterus, kidney and prostate as disclosed in US Pat. Nos. 7,238,785; 7517964 and 8084583, the Examples sections of which are incorporated herein by reference. The anti-hRS7 antibody is a humanized antibody that contains the CDR1 light chain complementarity determining region (CDR) sequences (KASQDVSIAVA, SEQ ID NO: 1); CDR2 (SASYRYT, SEQ ID NO: 2); and CDR3 (QQHYITPLT, SEQ ID NO: 3) and CDR1 heavy chain CDR sequences (NYGMN, SEQ ID NO: 4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO: 5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO: 6)

[0131] In a preferred embodiment, the antibodies used in the treatment of human diseases are human or humanized (CDR-grafted) variants of antibodies; although murine and chimeric versions of antibodies can be used. IgG molecules belonging to the same species as the delivery agents are generally preferred to minimize the immune response. This is especially important when considering retreatment. In humans, a human or humanized IgG antibody is less likely to elicit an anti-IgG immune response in patients. Antibodies such as hLL1 and hLL2 are rapidly internalized after binding to the internalizing antigen on the target cells, which also means that the transferred chemotherapeutic drug is rapidly internalized in the cells. However, antibodies having lower rates of internalization can also be used to provide selective therapy.

[0132] In another preferred embodiment, the therapeutic agent used in combination with the camptothecin conjugate of this invention may contain one or more isotopes. Radioactive isotopes useful for treating diseased tissues 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 therapeutic radionuclide preferably has a decay energy in the range of 20 to 6000 keV, preferably in the range of 60 to 200 keV for the Auger emitter, 100-2500 keV for the beta emitter, and 4000-6000 keV for the alpha emitter. The maximum decay energies of suitable beta emitting nuclides are preferably 20-5000 keV, more preferably 100-4000 keV, and most preferably 500-2500 keV. Also preferred are radionuclides which decay primarily to form 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 energies of suitable beta emitting nuclides are preferably less than 1000 keV, more preferably less than 100 keV, and most preferably less than 70 keV. Also preferred are radionuclides which decay primarily to form alpha particles. 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 energies of suitable alpha-emitting radionuclides are preferably 2000-10,000 keV, more preferably 3000-8000 keV, and most preferably 4000-7000 keV. Additional potentially useful radioisotopes 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 Br, ¹⁶⁹ Yb, etc.

[0133] Radionuclides and other metals can be delivered, for example, using chelating groups attached to the antibody or conjugate. Macrocyclic chelates such as NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and TETA (triethylenetetramine), are used with various metals and radiometals, especially with gallium, yttrium and copper radionuclides, respectively. Such metal chelate complexes can be made very stable by selecting the ring size for the metal of interest. Other ring chelates can be used such as macrocyclic polyethers to form complexes with ²²³ Ra.

[0134] Therapeutic agents for use in combination with the camptothecin conjugates described herein also include, for example, chemotherapeutic agents such as vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents, antibiotics, inhibitors 2, antimitotic agents, antiangiogenic and proapoptotic agents, in particular doxorubicin, methotrexate, taxol, other camptothecins, and others of these and other classes of antineoplastic agents, and the like. Other anti-cancer chemotherapy drugs include nitrogen mustard analogs, 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, 19th Ed. (Mack Publishing Co. 1995), and GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised editions of these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those skilled in the art.

[0135] Typical examples of useful drugs include, but are not limited to, 5-fluorouracil, afatinib, aplidine, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1 , busulfan, calicheamicin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizisphotini cytarabine, dacarbazine, dasatinib, dinatsiklib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolino (2P-DOX), tsianomorfolinodoksorubitsin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidofillotoksin, erlotinib, entinostat, agents that bind the estrogen receptor, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3 ', 5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl protein trans inhibitors ferases, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idealisib, ifosfamide, imatinib, L-asparaginase, lapatiniblazidamide, lecovolidine melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plikomycin, procarbazine-32765, PCI-pseudomixel, PSI-pI-346 , sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (aqueous DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uramustine (uracil mustard), vatalanibide vin, vinorelbine ZD1839. Such agents may be part of the conjugates described herein, or alternatively may be administered in combination with the described conjugates, either before, simultaneously with, or after the conjugate. Alternatively, one or more therapeutic naked antibodies known to those skilled in the art can be used in combination with the described conjugates. Typical examples of therapeutic naked antibodies are described above.

[0136] In preferred embodiments, the therapeutic agent for use in combination with a DNA-breaking antibody conjugate (eg, SN-38-ADC) is a microtubule inhibitor such as vinca alkaloid, taxanes, maytansinoid, or auristatin ... Typical examples of known microtubule inhibitors include paclitaxel, vincristine, vinblastine, mertansine, epothilone, docetaxel, discodermolide, combrestatin, podophyllotoxin, CI-980, phenylagistins, steganacins, curacins, 2-methoxyestradiolamines vinblastine, vinolluorin spongystatin, rhizoxin, tasidotin, halichondrins, hemiasterlins, cryptophycin 52, MMAE (monomethyluristatin E), and eribulin mesylate.

[0137] In an alternative preferred embodiment, the therapeutic agent for use in combination with a DNA degrading CAP, such as an SN-38 antibody conjugate, is a PARP inhibitor such as olaparib, talazoparib (BMN-673), rucaparib, veliparib, CEP 9722 , MK 4827, BGB-290, ABT-888, AG014699, BSI-201, CEP-8983 or 3-aminobenzamide.

[0138] In another alternative, a therapeutic agent used in combination with an 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 RN486.

[0139] In yet another alternative, the therapeutic agent used in combination with an antibody or immunoconjugate is a PI3K inhibitor such as idealisib, wortmannin, demethoxyviridine, perifosin, PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, palomid 529 (Palomid 529), GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL GNCUDE PI-907 , AEZS-136 or LY294002.

[0140] Therapeutic agents that can be used in combination with camptothecin conjugates can also include toxins conjugated to targeting moieties. Toxins that can be used include ricin, abrin, ribonuclease (RNase), DNase I, ranpyrnase, staphylococcal enterotoxin-A, antiviral protein of phytolacca american, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin, for example. , Pastan. Et al., Cell (1986) 47: 641, and Sharkey and Goldenberg, CA Cancer J Clin 2006 Jul-Aug; 56 (4): 226-43). Additional toxins suitable for use in this application are known to those skilled in the art and are disclosed in US Pat. No. 6,077,499.

[0141] Another class of therapeutic agents may include one or more immunomodulators. Suitable immunomodulators can be selected from cytokine, stem cell growth factor, lymphotoxin, hematopoietic factor, colony stimulating factor (CSF), interferon (IFN), erythropoietin, thrombopoietin, and a combination thereof. Especially useful are lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors such as interleukin (IL), colony stimulating factor such as granulocyte colony stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF), interferon such as interferons-α, -β, -γ or -λ, and a stem cell growth factor such as the designated "factor S1". Cytokines include growth hormones such as human growth hormone, N-methionyl-human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); hepatic growth factor; prostaglandin, a fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-α and -ß; Müllerian inhibiting substance; murine gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-ß; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-ß; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, -γ and -λ; colony stimulating factors (CSF) such as macrophage CSF (M-CSF); interleukins (IL) such as IL-1, IL-1α, 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 native sequence cytokines.

[0142] Useful chemokines include RANTES, MCAF, MIP1 alpha, MIP1 beta, and IP-10.

[0143] One of ordinary skill in the art will understand that immunoconjugates of this invention containing 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 , second immunoconjugate, radionuclide, toxin, drug, chemotherapeutic agent, radiation therapy, chemokine, cytokine, immunomodulator, enzyme, hormone, oligonucleotide, RNAi or siRNA. Such additional therapeutic agents can be administered alone, in combination with, or linked to the antibody-drug immunoconjugates of this invention.

Composition and introduction

[0144] Suitable routes of administration for conjugates include, but are not limited to, oral, parenteral, subcutaneous, rectal, transmucosal, intestinal, intramuscular, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, or intraglazal injection. The preferred routes of administration are parenteral. Alternatively, the compound can be administered locally rather than systemically, for example, by injecting the compound directly into a solid tumor.

[0145] Immunoconjugate compositions can be formulated according to known methods for preparing pharmaceutically acceptable compositions, the immunoconjugate being combined in admixture with a pharmaceutically acceptable excipient. One example of a pharmaceutically acceptable excipient is sterile phosphate buffered saline. 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, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990), and their revisions. publications.

[0146] In a preferred embodiment, the immunoconjugate composition is formulated in Goode's biological buffer (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 preferred buffers are MES or MOPS, preferably in the concentration range of 20 to 100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH 6.5. The composition may additionally contain 25 mM trehalose and 0.01% v / v. polysorbate 80 as excipients, with the final buffer concentration adjusted to 22.25 mM by addition of excipients. The preferred storage method is a lyophilized conjugate composition stored at temperatures ranging from minus 20 ° C to 2 ° C, with the most preferred storage at 2 ° C to 8 ° C.

[0147] The immunoconjugate composition can be formulated for intravenous administration, for example, by bolus injection, slow infusion, or continuous infusion. Preferably, the antibody of this invention is administered by infusion over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours. For example, the first 25-50 mg can be infused over 30 minutes, preferably even over 15 minutes, and the remainder is infused over the next 2-3 hours. Compositions for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain adjuvants such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable carrier, eg sterile, pyrogen-free water, prior to use.

[0148] Additional pharmaceutical methods can be used to control the duration of action of the therapeutic conjugate. Controlled release formulations can be prepared using polymers to complex or adsorb the immunoconjugate. For example, biocompatible polymers include poly (ethylene-co-vinyl acetate) matrices and polyanhydride copolymer matrices of stearic acid dimer 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 the immunoconjugate in 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 Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990), and their revised editions.

[0149] Generally, the dosage of administered immunoconjugate for humans will vary depending on factors such as the patient's age, weight, height, gender, general health, and previous medical history. It may be desirable to provide the recipient with a dose of the immunoconjugate that ranges from about 1 mg / kg to 24 mg / kg as a single intravenous infusion, although a lower or higher dosage may also be administered, depending on the circumstances. For example, the dosage of 1-20 mg / kg for a 70 kg patient is 70-1400 mg, or 41-824 mg / m ² to 1.7 m increase patient. The dosage may be repeated as necessary, e.g., once a week for 4-10 weeks, once a week for 8 weeks or once a week for 4 weeks. It can also be given less frequently, such as every two weeks for several months, or monthly or quarterly for many months if necessary for maintenance therapy. Preferred dosages may include, without limitation, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 22 mg / kg and 24 mg / kg. Any amount in the range from 1 to 24 mg / kg can be used. The dosage is preferably administered multiple times, once or twice a week. A minimum dosage regimen of 4 weeks, more preferably 8 weeks, more preferably 16 weeks or longer, can be used. The administration schedule may include administration once or twice a week, in a cycle selected from the group consisting of: (i) weekly; (ii) every two weeks; (iii) one week of therapy followed by two, three, or four weeks off; (iv) two weeks of therapy followed by one, two, three or four weeks off; (v) three weeks of therapy followed by one, two, three, four or five weeks off; (vi) four weeks of therapy followed by one, two, three, four or five weeks off; (vii) five weeks of therapy followed by one, two, three, four or five weeks off; and (viii) monthly. The cycle can be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.

[0150] Alternatively, the immunoconjugate can be administered as a single dose every 2 or 3 weeks, repeating a total of at least 3 doses. Or twice a week for 4-6 weeks. If the dosage is lowered to approximately 200-300 mg / m ² (340 mg per dose for the patient increase 1.7 m, or 4.9 mg / kg for a 70 kg patient), it may be administered once or twice a week for 4 -10 weeks. Alternatively, the frequency of dosing can be reduced, namely, every 2 or 3 weeks for 2-3 months. It has been found, however, that even higher doses, such as 12 mg / kg once a week or once every 2-3 weeks, can be administered by slow intravenous infusion, with repeated dosing cycles. The dosing schedule can be repeated at different intervals if desired, and the dosage can be administered by various parenteral routes, with appropriate dose and schedule adjustments.

[0151] In preferred embodiments, the immunoconjugates are useful in cancer therapy. Examples of cancers include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma and leukemia, myeloma, or lymphoid malignancies. More specific examples of such cancers are listed below and include: squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), Ewing's sarcoma, Wilms tumor, astrocytomas, lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell lung cancer, cancer peritoneum, gastric or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors, medullary thyroid cancer , differentiated thyroid cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, anal cancer, cancer penis; and head and neck cancer. The term "cancer" includes primary malignant cells or tumors (for example, those whose cells have not migrated to areas of the subject's body other than the site of the original malignant neoplasm or tumor) and secondary malignant cells or tumors (for example, those resulting from metastasis, migration of malignant cells or tumor cells to secondary sites other than the original tumor site).

[0152] Other examples of cancers or malignant neoplasms include, but are not limited to: childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical cancer, (primary) adult hepatocellular cancer, (primary) adult liver cancer, acute adult lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-associated lymphomas, AIDS-associated malignant neoplasms, anal cancer, astrocytoma, bile duct cancer , bladder cancer, bone cancer, brainstem glioma, brain tumors, breast cancer, renal pelvis and ureter cancer, central nervous system lymphoma (primary), central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, children (primary ny) hepatocellular cancer, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, brain stem glioma in children, pediatric cerebellar astrocytoma, cerebral astrocytoma in children, extracranial germ cell tumors in children, Hodgkin's disease in children, Hodgkin's lymphoma in children, glioma of the hypothalamus and optic tract in children, lymphoblastic leukemia in children, medulloblastoma in children, childhood non-Hodgkin's lymphoma, pediatric pineal and supratentorial primitive neuroectodermal tumors, childhood primary liver cancer, rhabdomyosarcoma in children, soft tissue sarcoma in children glioma of the optic tract and hypothalamus, 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 gland cancer , tumor extracranial germ cells, extragonal germ cell tumor, extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher disease, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal tract tumors, germ cell tumors, hairy cell leukemia , head and neck cancer, hepatocellular cancer, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, bowel cancers, intraocular melanoma, islet cell cancer, pancreatic islet cell cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, cancer liver, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic asymptomatic primary squamous cell carcinoma of the neck, metastatic primary squamous cell carcinoma of the neck, multiple myeloma / myeloma cells, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, asymptomatic cancer primary metastatic cervical cancer / malignant fibrous sarcoma, osteosarcoma / malignant fibrous histiocytoma, osteosarcoma / malignant fibrous bone histiocytoma, epithelial ovarian cancer, germ cell tumor of the ovaries, potential low-grade ovarian tumor, pancreatic cancer of the oxychromic gland, paraprotective cancer , pituitary tumor, primary lymphoma of the central nervous system, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoidosis, sarcomas, syndrome Sesari rum, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma of the neck, stomach cancer, supratentorial primitive neuroectodermal and pineal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, cancer of the transitional epithelium of the renal pelvis and ureter, trophoblastic tumors, cancer of the cells of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, glioma of the optic tract and hypothalamus, vulvar cancer, macroglobulinemia, Waldenstremil's tumor and any other lymphoproliferative disease, in addition to neoplasia, located in the above organ systems.

[0153] The methods and compositions described and claimed herein can be used to treat malignant or precancerous conditions and to prevent progression to a neoplastic or malignant condition, including, without limitation, the disorders described above. Such uses are indicated in conditions with known or suspected pre-existing progression to neoplasia or cancer, in particular where non-tumor cell growth has occurred, including hyperplasia, metaplasia, or especially dysplasia (for a review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., WBSaunders Co., Philadelphia, pp. 68-79 (1976)).

[0154] Dysplasia is often a precursor to cancer and occurs mainly in the epithelium. This is the most disordered form of growth of non-tumor cells associated with the loss of the uniformity of individual cells and the architectural orientation of cells. Dysplasia usually occurs where chronic irritation or inflammation exists. Dysplastic disorders that can be treated include, but are not limited to, anhydrotic ectodermal dysplasia, anterofacial dysplasia, asphyxial thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cranial dysplasia kraniodiafizarnuyu dysplasia kraniokarpotarzalnuyu dysplasia kraniometafizarnuyu dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, dysplasia enamels, encephalo-ophthalmic dysplasia, tarzomegaliyu, metaphyseal multiple dysplasia, epiphyseal dot dysplasia, epithelial dysplasia, fatsiodigitogenitalnuyu dysplasia, familial fibrous dysplasia of jaws, familial spongy white nevus of the mucous membrane, fibromuscular dysplasia, fibrous bone dysplasia, cement dysplasia, hereditary renal retinal dysplasia, hydrotic ectodermal dysplasia, hyp ohydrotic ectodermal dysplasia, lymphopenic thymus dysplasia, breast dysplasia, maxillofacial osteodysplasia, metaphyseal dysplasia, Mondini dysplasia, monoosal fibrous dysplasia, mucoepithelial dysplasia, multiple epiphyseal-ocular dysplasia, ocular dysplasia oculomaxillary dysplasia, periapical cement dysplasia, polyosseous fibrous dysplasia, pseudoachondroplastic spondyloepiphyseal dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepiphyseal dysplasia, and ventriculoradial dysplasia.

[0155] Additional precancerous disorders that can be treated include, but are not limited to, benign dysproliferative disorders (eg, benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, diamond-shaped skin of the neck (Farmer's Skin), solar cheilitis and solar keratosis.

[0156] In preferred embodiments, the method of the invention is used to inhibit the growth, progression and / or metastasis of cancers such as those listed above.

[0157] Additional hyperproliferative diseases, disorders and / or conditions include, without limitation, progression and / or metastases of malignant tumors and related disorders such as leukemia (including acute leukemias; for example, acute lymphocytic leukemia, acute myelocytic leukemia [including myeloblastic, promyelocytic , myelomonocytic, monocytic, and erythroleukemia]) and chronic leukemias (eg, chronic myelocytic leukemia [granulocytic] and chronic lymphocytic leukemia), polycythemia vera, lymphomas (eg, Hodgkin's disease and non-Hodgkin's lymphoma), multiple myelogenous leukemia, macroglobuline suthema , and solid tumors, including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myyxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, leucoma endothelioma Oma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary bronchogenic carcinoma carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonic carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, urinary bladder cancer, epithelial carcinoma, glioma, astrocytoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma (emangioblastoma), acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

[0158] Autoimmune diseases that can be treated with immunoconjugates may include acute and chronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea, severe pseudoparalytic myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic diabetic fever, polyglandupea Genoch-Schönlein, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, exudative erythema multiforme, IgA-nephropathy syndrome thromboangiitis, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, bullous pemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranous sclerosus tabes of the spinal cord, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis or fibrosing alveolitis.

Sets

[0159] Various embodiments may relate to kits containing components useful for treating cancer in a patient. Typical examples of kits may contain at least one antibody conjugated to a drug as described herein. If the composition containing the components for administration is not formulated for delivery via the digestive tract, for example, by oral delivery, a device capable of delivering the components of the kit in some other way may be included. One typical device for applications such as parenteral delivery is a syringe that is used to inject the composition into a subject by injection. Inhalation devices can also be used.

[0160] The components of the kit can be packaged together or divided into two or more containers. In some embodiments, the containers can be vials that contain sterile lyophilized formulations of the composition suitable for reconstitution. The kit can also contain one or more buffers suitable for diluting and / or diluting other reagents. Other containers that can be used include, but are not limited to, a pouch, tray, box, tube, and the like. Kit components can be packaged and stored sterile in containers. Another component that may be included are instructions for the person using the kit to use it.

Examples of

[0161] Various embodiments of the present invention are illustrated by the following non-limiting examples.

Example 1. Clinical Trials of Sacituzumab Govitecan (IMMU-132) in Metastatic Urothelial Cancer

[0162] There are no FDA approved treatments for patients with metastatic platinum-resistant urothelial carcinoma (PRUC). The percentage of patients with an objective response to second-line chemotherapy was usually less than 20%, with a median overall survival of less than 1 year. We report in this document our experience with 6 previously intensively treated patients with advanced PRUC (ClinicalTrials ID NCT01631552) using a novel antibody-drug conjugate, sacituzumab govitecan (IMMU-132). This antibody-drug conjugate contains the active metabolite of irinotecan, SN-38, conjugated to an anti-Trop-2 antibody (hRS7).

[0163] Trop-2 is widely expressed in less than or equal to 83% of urothelial carcinomas. Of the 6 patients, 3 had a clinically significant response (progression-free survival 6.7 to 8.2 months; overall survival 7.5+ to 11.4+ months). Sacituzumab Govitecan is well tolerated. Based on these results, phase II trials were initiated. This report demonstrates the utility of anti-Trop-2 antibody-drug conjugates such as sacituzumab govitecan as a novel therapeutic strategy for the treatment of PRUC.

Introduction

[0164] Urothelial carcinoma of the bladder (UC) is the sixth most common cancer (eg, Sharma et al., 2009, Am Fam Physician 80: 717-23). Cisplatin-based combination chemotherapy is the only known treatment that has shown a beneficial effect on survival for patients with advanced disease (Logothetis et al., 1990, J Clin Oncol 8: 1050-55; Loehrer et al., 1992, J Clin Oncol 10 : 1066-73). However, only a small subpopulation achieves long-term survival. For clinical trial participants, median overall survival was 15 months and 5-year survival was only 15% (von der Maase et al., 2005, J Clin Oncol 23: 4602-8) After progression 6 to 12 months after chemotherapy (platinum-resistant urothelial carcinoma [PRUC]), regardless of perioperative or advanced setting, survival was only 4 to 9 months for eligible subjects. No second-line chemotherapeutic agents have been approved in the United States, and only vinflunine is available in Europe (Bellmunt et al., 2009, J Clin Oncol 27: 4454-61) The development of effective second-line treatments for advanced urothelial cancer is an important unmet medical need ( Faltas et al., 2015, Expert Opin Ther Targets 19: 515-25).

[0165] Antibody-drug conjugates (ADCs) targeting cell surface antigens represent an attractive therapeutic strategy for chemotherapy-resistant tumors, including PRUC (Cardillo et al., 2015, Bioconjug Chem 26: 919-31). Sacituzumab govitecan (IMMU-132) is a second generation CAP containing a humanized anti-Trop-2 monoclonal antibody (hRS7) conjugated to the active metabolite of irinotecan, SN-38 (Goldenberg et al., 2015, Oncotarget 6: 22496-5120). It has demonstrated acceptable toxicity and excellent therapeutic activity against several solid tumors in both preclinical and clinical settings (Cardillo et al., 2015, Bioconjug Chem 26: 919-31; Starodub et al., 2015, Clin Cancer Res 21: 3870-78), and is a rational choice for targeting UC. It is known that the Trop-2 (TACSTD2) protein is expressed in normal urothelium (Stepan et al., 2011, J Histochem Cytochem 59: 701-10) and is less than or equal to 83% of urothelial carcinomas (Faltas et al., 2016, Clin Genitourin Cancer 14: e75-9). Phase II clinical trials using irinotecan in patients with PRUC showed a total treatment efficacy of only 5% (95% CI, 1% -17%), including a 33-month complete response and an overall survival of 5.4 months (Beer et al., 2008, Clin Genitourin Cancer 6: 36-9). Irinotecan has also been used in combination with other drugs (Chaudhary et al., 2014, Am J Clin Oncol 37: 188-93).

[0166] In an expanded study evaluating sacituzumab govitecan (ClinicalTrials ID NCT01631552), we initially studied 6 patients with PRUC, 3 of whom achieved clinically significant responses. This Example describes this clinical experience, which demonstrates that this CAP is an attractive candidate for the treatment of PRUC.

Materials and methods

[0167] Humanized anti-Trop-2 antibody RS7 (hRS7) was prepared as described in US Pat. No. 7,238,785, the Figures and Examples section of which are incorporated herein by reference. SN-38 attached to a CL2A linker was prepared and conjugated with hRS7 (anti-Trop-2) in accordance with US patent No. 7999083 (Examples 10 and 12 of which are incorporated herein by reference). The conjugation protocol resulted in a ratio of about 6-8 attached SN-38 molecules per antibody molecule.

[0168] Patients were eligible for clinical trials using IMMU-132 if they had advanced PRUC, performance status 0 to 1 on the Eastern Cooperative Oncology Group scale, and intact organ function ( Starodub et al., 2015, Clin Cancer Res 21: 3870-78). All patients gave informed consent. All experimental procedures were carried out in accordance with the approved methodological recommendations. Sacituzumab govitecan was administered intravenously on days 1 and 8 of 21-day cycles, which were repeated until the development of dose-limiting toxicity or progression. The response was assessed using the Response Evaluation Criteria in Solid Tumors, version 1.1. Whenever possible, immunohistochemical staining of biopsy samples from archival tumors obtained from treated patients was performed as previously described (Starodub et al., 2015, Clin Cancer Res 21: 3870-78).

results

[0169] The median age of the patients was 72.5 years (range, 42 to 80 years). All patients had metastatic disease and had previously received treatment with platinum-containing regimens and other lines of therapy (median number of previous therapies - 3; Table 2). Of the 6 patients, 5 belonged to low or intermediate risk groups according to the predictive model for UC patients receiving systemic salvage therapy (Sonpavde et al., 2015, J Clin Oncol 33 (abstract 311). All 6 patients with PRUC were available Two achieved a partial response, with the patient with the best therapeutic effect showing a 38% decrease in target lesions, including liver metastases (Fig. 1). One patient had stable disease with a 28% decrease in target lesions, and 3 of patients had progressive disease, of which 1 patient was diagnosed with progressive disease according to the criteria for evaluating solid tumor response, version 1.1, due to a new lesion, despite a 12% decrease in its target lesions during treatment (Table 2). of patients with a clinically significant response, progression-free survival ranged from 6.7 to 8.2 months, and overall survival ranged from 7.5+ to 11.4+ months.

[0170] Sacituzumab-govitecan was generally well tolerated. Two patients had grade 3 toxicity (flank pain and bacteremia). No grade 4 non-hematological toxicity was observed. Immunohistochemical analysis of archived PRUC tumor tissue from patients treated with sacituzumab govitecan showed significant expression of the Trop-2 protein on the cell surface (Fig. 2).

Table 2. Baseline characteristics and clinical results.

PC.№ Floor Age (years) Primary lesion UC Areas of metastasis Prior treatment GB (g / dl) LDH (IU / L) Albumin (g / dl) Sacituzumab Govitecan Best answer (%) PFS (months) OV (months) Dose (mg / kg) Cycles (n) 1 M 42 bladder bone gemcitabine + carboplatin 13 134 3.8 12 4 PZ (−30) 1.5 10.8 gemcitabine + cisplatin docetaxel 2 M 80 pelvis liver, lungs, mediastinal lymph nodes gemcitabine + carboplatin 12.7 210 4 ten 6 PZ (−30) 1.9 1.9+ 3 M 71 bladder pelvic, para-aortic, RPLNs paclitaxel, ifosfamide, cisplatin 11.1 218 3.9 eight 4 PZ (−12) 1.8 1.8+ docetaxel 4 M 66 pelvis Lungs, para-aortic lymph nodes, psoas muscle gemcitabine + cisplatin 9.6 148 3.6 eight 13 NW (−28) 6,7 11.4+ 5 M 74 bladder SCV, mediastinal, RPLNs BCG 13.5 156 4.4 eleven 12+ PZ (−33) 7.5+ 7.5+ gemcitabine + cisplatin Research (DN24-02) 6 M 75 ureter liver, lungs, pelvic lymph nodes, peritoneum gemcitabine + cisplatin (adjuvant) 9.3 187 2.6 eight 25 CHO (−38) 8.2 10.5 gemcitabine + carboplatin docetaxel + icrucumab

Abbreviations: BCG is the bacillus Calmette-Guerin; GB is hemoglobin; LDH is lactate dehydrogenase; M is male; OS is overall survival; PZ is a progressive disease; PFS is progression-free survival; CHO represents a partial response; PC. No. is the patient number; RECIST 1.1 is a Response Evaluation Criteria in Solid Tumors, version 1.1; RPLNs are retroperitoneal lymph nodes; SCV is supraclavicular; SD is a stable disease; UC is urothelial carcinoma.

Discussion

[0171] Although the vinca alkaloid vinflunine is available in Europe due to results from phase III trials comparing it to the best second-line maintenance treatment, its efficacy was marginal, with no improvement in overall survival (Bellmunt et al., 2009, J Clin Oncol 27: 4454 -61). The overall therapy efficacy for patients receiving second-line therapy such as vinflunine or other agents, including taxanes and pemetrexed, was typically less than 20%, with a median overall survival of only 4 to 9 months (Bellmunt et al., 2009, J Clin Oncol 27: 4454-61; Sweeney et al., 2006, J Clin Oncol 24: 3451-57; Galsky et al., 2007, Invest New Drugs 25: 265-70). A recently presented positive randomized phase II trial of docetaxel with or without ramucirumab or icrucumab demonstrated a 5% efficacy and 10.4 weeks progression-free survival in a control group treated with docetaxel alone (Petrylak et al., 2012, J Clin Oncol 30 (Abstract TPS4675) A large institutional review of the commonly prescribed second-line drug, pemetrexed, showed an objective response rate of 5% (95% CI, 1% -9%) and a median progression-free survival of 2.4 months (Bambury et al. , 2015, Oncologist 20: 50-15) Thus, at this time, patients with PRUC have a limited choice of therapy.

[0172] In this first group of PRUC patients enrolled in Phase I / II trials, sacituzumab govitecan showed a preliminary signal of significant clinical activity in this intensive previous treatment cohort. As previously observed for UC cell lines and patient-derived PRUC tumors, we detected high levels of Trop-2 protein expression in tumor biopsies from patients receiving sacituzumab govitecan (Fig. 2). Our sample size did not allow for a correlation between Trop-2 expression levels and clinical response. However, the activity observed in this small group of PRUC patients, with good overall tolerance, is consistent with preclinical results indicating that PAP selectively delivers a significant proportion of the potent drug to tumor cells rather than normal cells (Sharkey et al. , 2015, Clin Cancer Res 21: 3870-78). The above data demonstrate the safety and efficacy of IMMU-132 in metastatic urothelial cancer.

Example 2. Further studies of IMMU-132 in metastatic urothelial cancer

[0173] Following example 1, conducted further studies in patients with mUC (metastatic urothelial cancer), previously receiving platinum-containing chemotherapy. These patients have limited therapeutic options, with a minority of patients responding to checkpoint inhibitor (IO) immunotherapy. We present additional evidence for the safety and activity of sacituzumab govitecan (IMMU-132) as therapy for mUC patients previously treated with chemotherapy (ClinicalTrials.gov, NCT01631552).

Method

[0174] We enrolled 32 patients with mUC and ECOG PS 0-1 (functional status 0 to 1 on the Eastern Cooperative Cancer Group) who did not respond to one or more previous standard therapies (median = 3; range, 1-5 IMMU-132 was administered at a dose of 8 or 10 mg / kg on days 1 and 8 every 21 days, continuing treatment until disease progression (PD) or unacceptable toxicity. Patients with assessed response received greater than or equal to 2 doses, and had greater than or equal to 1 response score after baseline.

results

[0175] Twenty-five patients [mean age 68 years (range: 50-91), 24 men] were evaluated to determine the safety and efficacy of treatment; 23 received prior platinum therapy; 46% have had more than 2 previous therapies; 4 also received IO agents. The foci of metastasis included liver (N = 4; 16%), lungs (N = 7; 28%), bones (N = 4; 16%) and lymph nodes (N = 16; 64%). Patients received an average of 7 cycles (range, 2-23) of IMMU-132. The percentage of patients with an objective response (ORR) was 36% (9/25) [1 complete (CR) and 8 partial responses (OR)]; 44% (11/25) had stable disease (SD). In addition, patients with 1-line prior chemotherapy had an objective response rate (ORR) of 53.8% (7/13) and 16.7% in patients with 2-5 lines of prior therapy. The median PFS for all patients is 7.2 months. (95% CI, 4.9-10.7); median survival has not yet been reached. Of 4 patients with progression after previous IO, 1 experienced OR and 2 had CV with IMMU-132. The duration of response in CR / PR patients at this time is 5.1 months. (95% CI, 4.1-12.9) and 10/11 patients (5 s greater than or equal to 20% tumor shrinkage) had stable disease for more than 4 months. Grade 4 neutropenia (16%) lasted less than 7 days, and grade 3 nonhematologic adverse effects (AE) included fatigue (12%) and hypophosphatemia (8%). There were no treatment-related deaths. Analysis of Trop-2 expression revealed positive staining from 1+ to 3+ in 95% of 19 archived patient samples.

Conclusion

[0176] With an objective response rate (ORR) of 36% and a median PFS of 7.2 months in a previously intensively treated population, these intermediate results show the efficacy and tolerability of IMMU-132 as a ^2nd- line therapy or later in patients with mUC who have received prior treatment with platinum or IO.

Example 3. Clinical trials of anti-Trop2 IMMU-132 CAP in various Trop-2 + cancers

Summary

[0177] This example provides the results of a phase I clinical trial and an ongoing phase II continuation using IMMU-132, an PAP internalizing humanized anti-Trop-2 hRS7 antibody conjugated with a pH-sensitive linker to SN-38 (mean drug-antibody ratio = 7.6). Trop-2 is a type I calcium transmembrane protein expressed at high density (about 1 × 10 ⁵ ), frequency and specificity in many human carcinomas, with limited expression in normal tissue. Preclinical studies in nude mice carrying Capan-1 human pancreatic tumor xenografts have shown that IMMU-132 is capable of delivering up to 120 times more SN-38 to the tumor than with maximally tolerated irinotecan therapy.

[0178] The results shown were from an initial phase I trial in 25 patients with multiple prior therapy failure (some of which included drugs that inhibit topoisomerase I / II) and an ongoing phase II continuation, currently reporting 69 patients, in including colorectal cancer (CRC), small cell and non-small cell lung cancer (SCLC, NSCLC, respectively), triple negative breast cancer (TNBC), pancreatic cancer (PC (PDC - other pancreatic duct carcinoma)), esophageal and esophageal cancer types of cancer.

[0179] As discussed in detail below, Trop-2 was not detected in serum, but was strongly expressed (immunohistochemical staining greater than or equal to 2 ⁺ ) in most archival tumors. In the 3 + 3 trials, IMMU-132 was administered on days 1 and 8 in repeated 21-day cycles, starting at 8 mg / kg / dose and then 12 and 18 mg / kg, to dose-limiting neutropenia. To optimize cumulative treatment with minimal delays, phase II focuses at 8 and 10 mg / kg (n = 30 and 14, respectively). Of the 49 patients who reported associated side effects (PE) at this time, neutropenia greater than or equal to grade 3 was observed in 28% (4% - grade 4). The most common non-hematological toxicities in these patients were fatigue (55%; ≥G3 = 9%), nausea (53%; ≥G3 = 0%), diarrhea (47%; ≥G3 = 9%), alopecia (40%) and vomiting (32%; ≥G3 = 2%); alopecia was also common. Homozygous UGT1A1 * 28 / * 28 was found in 6 patients, 2 of whom had more severe hematological and gastrointestinal toxicities.

[0180] In Phase I and in the continuation phase, 48 patients are currently participating (excluding PCa) who were assessed by RECIST / CT for best response. Seven (15%) of the patients had a partial response (PR), including patients with CRC (N = 1), TNBC (N = 2), SCLC (N = 2), NSCLC (N = 1), and esophageal cancer (N = 1), and another 27 patients (56%) had stable disease (SD), a total of 38 patients (79%) with a disease response; Eight of 13 CT-assessed PCa patients (62%) had SD, with a median time to progression (TTP) of 12.7 weeks, compared with 8.0 weeks in their last prior therapy. The URT for the remaining 48 patients was 12.6+ weeks (range 6.0 to 51.4 weeks). Plasma CEA (cancer embryonic antigen) and CA19-9 correlated with responses (from patients) who had elevated blood titers of these antigens. Anti-hRS7 or anti-SN-38 antibodies were not detected despite the dosage for several months.

[0181] The conjugate was cleared from serum within 3 days, which is consistent with in vivo animal studies in which 50% SN-38 was released daily, with more than 95% of SN-38 in serum bound to IgG in non-glucuronidated form, and at concentrations reported to be up to 100 times the SN-38 levels in patients treated with irinotecan. These results indicate that HRS containing hRS7-SN-38 is therapeutically active in metastatic solid cancers with manageable diarrhea and neutropenia.

Pharmacokinetics

[0182] Two ELISA methods were used to measure IgG clearance (capture by anti-hRS7 idiotypic antibody) and intact conjugate (capture by anti-SN-38 IgG / probe with anti-hRS7 idiotypic antibody). SN-38 was measured by method HPLC. Clearance of the total IMMU-132 fraction (intact conjugate) was faster than IgG (not shown), reflecting the known gradual release of SN-38 from the conjugate. Determination of SN-38 (unbound and total) by HPLC showed that more than 95% of SN-38 in serum was associated with IgG. Low SN-38G concentrations suggest that IgG-associated SN-38 is protected from glucuronidation. Comparison of ELISA results for conjugate and HPLC for SN-38 showed that they overlap, indicating that ELISA can be used instead of monitoring SN-38 clearance.

[0183] A summary of the dosing regimen and patient pool is given in Table 3.

Table 3. Parameters of clinical trials

Dosing scheme Injection once a week for 2 weeks every 21 days for up to 8 cycles. At initial enrollment, the planned dose was delayed and the dose was reduced in the event of treatment-related toxicity greater than or equal to grade 2; the protocol was changed taking into account the delay and dose reduction only in the case of toxicity greater than or equal to grade 3. Dose level cohorts 8, 12, 18 mg / kg; later reduced to an intermediate dose level of 10 mg / kg. Cohort size Phase I standard circuit [3 + 3]; the follow-up included about 15 patients with selected cancers. DLT (dose-limiting toxicity) ANC (absolute neutrophil count) grade 4 greater than or equal to 7 days; febrile neutropenia of any grade duration greater than or equal to 3; platelets grade 4 (G4 Plt) greater than or equal to 5 days; hemoglobin grade 4; nausea / vomiting / diarrhea (N / V / D) grade 4 of any duration / nausea / vomiting / diarrhea grade 3 for more than 48 hours; grade 3 infusion reactions; associated non-hematological toxicity grade greater than or equal to 3. Maximum Allowable Dose (DMD) The maximum dose at which more than or equal to 2/6 of patients tolerate the ^1st 21-day cycle without delay or dose reduction or toxicity grade ≥G3. Patients Metastatic colorectal, pancreas, stomach, esophagus, lung (NSCLC, SCLC), triple negative breast (TNBC), prostate, ovary, kidney, bladder, head / neck, hepatocellular. Refractory / relapsing after standard treatment regimens for metastatic cancer. Admission does NOT require prior irinotecan-containing therapy. Absence of volumetric lesions more than 5 cm.
At least 4 weeks after any "major" surgery, and 2 weeks after radiation or chemotherapy regimens. Gilbert's disease or known metastatic lesion of the central nervous system is excluded.

Clinical Trials Status

[0184] A total of 69 patients (including 25 patients in phase I) with various metastatic cancers have been described with an average of 3 previous therapies. Eight patients had clinical progression and were excluded prior to CT. Thirteen patients with pancreatic cancer amenable to CT assessment were enrolled separately. The median URT (time to progression) in patients with PCa (pancreatic cancer) was 11.9 weeks (range, 2 to 21.4 weeks), compared with a median URT of 8 weeks for the last prior therapy.

[0185] A total of 48 patients with various cancers underwent at least 1 CT scan, which determined the best response (Fig. 3) and time to progression (URT; not shown). Pooled data on the best response - of 8 evaluated patients with TNBC (triple negative breast cancer), 2 had OR (partial response), 4 - CV (stable disease) and 2 - PZ (progressive disease), with a total response [HR + SZ] 6/8 (75%). For SCLC (small cell lung cancer), 4 patients assessed had 2 RR, 0 CZ and 2 PZ, with a total response of 2/4 (50%). For colorectal cancer (colorectal cancer), 18 patients assessed had 1 RR, 11 SD, and 6 PZ, with a cumulative response of 12/18 (67%). For esophageal cancer, 4 patients evaluated had 1 RR, 2 SD, and 1 PZ, with a total response of 3/4 (75%). For NSCLC (non-small cell lung cancer), 5 patients assessed had 1 RR, 3 CZ and 1 PZ, with a total response of 4/5 (80%). For all treated patients, 48 patients assessed had 7 RR, 27 SD, and 14 RR, with a total response of 34/48 (71%). These results demonstrate that anti-Trop2 CAP (hRS7-SN-38) shows significant clinical efficacy against a wide range of solid tumors in humans.

[0186] The reported side effects of therapy (unwanted effects) are summarized in Table 4. As can be seen from the data shown in Table 4, the therapeutic efficacy of hRS7-SN-38 was achieved at doses of CAP showing an acceptably low level of adverse side effects.

Table 4.

List of Associated Side Effects for IMMU-132-01
Criteria: Total greater than or equal to 10% or greater than or equal to 3 degrees N = 47 patients TOTAL Degree 3 Degree 4 Fatigue 55% 4 (9%) 0 Nausea 53% 0 0 Diarrhea 47% 4 (9%) 0 Neutropenia 43% 11 (24%) 2 (4%) Alopecia 40% - - Vomit 32% 12 %) 0 Anemia 13 % 2 (4%) 0 Dysgeusia 15 % 0 0 Pyrexia 13 % 0 0 Abdominal pain eleven % 0 0 Hypokalemia eleven % 12 %) 0 Decreased white blood cell count (WBC) 6% 12 %) 0 Febrile neutropenia 6% 12 %) 2 (4%) Deep vein thrombosis 2% 12 %) 0 CTCAE (Common Adverse Event Criteria Terminology) Assessment v 4.0

[0187] The studies described in Table 4 were continued, to date, 261 patients have been enrolled. The results (not shown) are broadly in line with the trends shown in Table 4, with the exception of neutropenia alone, showing a frequency of Grade 3 or higher adverse effects of more than 10% of patients examined. For all other adverse events, the response rate of grade 3 or higher was less than 10%. This distinguishes the immunoconjugates of this invention from the vast majority of CAPs and, in some embodiments, the inventive methods and compositions relate to anti-Trop-2 CAPs, which are effective in a variety of solid tumors, with a frequency of grade 3 or higher adverse events of less than 10%. patients for all adverse events except neutropenia. With continued studies, for a total of 421 samples from 121 patients with available baseline samples and at least one follow-up sample, no anti-hRS7 or anti-SN-38 antibodies were detected despite repeated treatment cycles.

[0188] Typical examples of partial responses to anti-Trop-2 CAP were confirmed by CT data (not shown). As a typical example of OR in CRC, a 62-year-old woman with a primary diagnosis of CRC underwent primary hemicolectomy. Four months later, she underwent liver resection for liver metastases and received FOLFOX for 7 months and 5FU for 1 month. At the time of enrollment, she had multiple lesions, mainly in the liver (3+ Trop-2 by immunohistology), and was prescribed hRS7-SN-38 at an initial dose of 8 mg / kg approximately 1 year after the initial diagnosis. On her first CT scan, OR was achieved, with a 37% reduction in target lesions (not shown). The patient continued treatment, reaching a maximum reduction of 65% after 10 months of treatment (not shown), with a decrease in CEA from 781 ng / ml to 26.5 ng / ml), followed by progression after 3 months.

[0189] As a typical example of NSCLC in NSCLC, a 65-year-old man was diagnosed with stage IIIB (squamous cell) NSCLC. Initial treatment with caboplatin / etoposide (3 months) combined with X-ray therapy (XRT) 7000 cGy resulted in a response of 10 months. He was then prescribed supportive Tarceva therapy, which he continued until he was asked to participate in the IMMU-132 trial, in addition to having a lumbar laminectomy. He received the first dose of IMMU-132 after 5 months of Tarcev, while having a 5.6 cm lesion in the right lung with copious pleural effusion. Immediately after the administration of doses he is 6 ^minutes two months first CT scan showed that the primary target lesion decreased to 3.2 cm (not shown).

[0190] As a typical example of OR in SCLC, a 65-year-old woman was diagnosed with poorly differentiated SCLC. After taking carboplatin / etoposide (a topoisomerase II inhibitor) that ended 2 months later with no response, and then topotecan (a topoisomerase I inhibitor) that ended 2 months later, also without a response, she received topical radiotherapy (XRT) (3000 cGy ), which ended after 1 month. However, by the next month, the progression continued. The patient started IMMU-132 from the next month (12 mg / kg; reduced to 6.8 mg / kg; Trop-2 3+ expression), and after two months of IMMU-132 there was a 38% decrease in target lesions, including a significant decrease the main focus in the lungs (not shown). The patient progressed 3 months after taking 12 doses.

[0191] These results are important in that they demonstrate the effectiveness of anti-Trop-2 CAP, even in patients with therapy failure or disease progression after multiple prior therapies.

In conclusion, at the doses used, the primary toxicity was controlled neutropenia, with several cases of grade 3 toxicity IMMU-132 demonstrated evidence of activity (OR and long-term SD) in patients with recurrent / refractory triple negative breast cancer, small cell lung cancer, non-small cell cancer lung, colorectal cancer and esophageal cancer, including patients with a previous history of relapse after therapy with a topoisomerase-I inhibitor. These results demonstrate the efficacy of anti-Trop-2 CAP for a wide range of cancers resistant to existing therapies.

Example 4. Treatment of patients with advanced metastatic pancreatic cancer using anti-Trop2 CAP

Summary

[0192] Trop-2 is a type I transmembrane calcium-transducing protein expressed with high density, frequency and specificity in many epithelial cancers, including pancreatic duct adenocarcinoma, with limited expression in normal tissue. All 29 pancreatic tumor microarray samples tested were Trop-2 positive for immunohistochemistry, and human pancreatic cancer cell lines were found to express 115-891 thousand copies of Trop-2 on the cell membrane.

[0193] Above, we described the results of the IMMU-132 phase I studies, including patients with 13 different types of tumors, using the 3 + 3 scheme. The dose-limiting phase I toxicity was neutropenia. More than 80% of the 24 patients evaluated in these studies had long-term stable disease, with partial responses (RECIST) seen in patients with colorectal cancer (CRC), triple-negative breast cancer (TNBC), small cell and non-small cell lung cancer (SCLC, NSCLC) ) and esophageal cancer (EAC (EAC - esophageal adenocarcinoma)). This example presents the results of IMMU-132 Phase I / II studies in a cohort of patients with metastatic PCa. PCa patients with an average of 2 prior therapy failures (range 1-5) received IMMU-132 on days 1 and 8 of repetitive 21-day cycles.

[0194] In a subgroup of PCa patients (N = 15), 14 received prior treatment regimens that included gemcitabine. Baseline toxicity data in 9 patients included neutropenia [3 of 9, greater than or equal to grade 3, 33%; and 1 case of grade 4 febrile neutropenia), which resulted in delay or dose reduction. Two patients had grade 3 diarrhea; none of the patients had grade 3-4 nausea or vomiting. Alopecia (grade 1-2) was observed in 5 out of 9 patients. The best response was assessed in 13 of 14 patients, with 8 cases of stable illness ranging from 8 to 21.4 weeks (median 12.7 weeks; 11.9 weeks for all 14 patients). One patient who is continuing treatment has not yet had the first CT scan. Five patients had progressive disease according to RECIST; 1 dropped out after only 1 dose due to clinical progression and was not evaluated. Serum CA19-9 titers decreased in 3 patients with stable disease by 23% to 72%. Despite repeated administrations, none of the patients developed an antibody response to IMMU-132 or SN-38. Serum samples with peak and trough drug concentrations showed that the clearance of IMMU-132 was faster than IgG, which was expected based on the known local release of SN-38 in the tumor cell. Concentrations of SN-38 bound to IgG in peak samples from one patient treated with 12 mg / kg IMMU-132 showed levels of about 4000 ng / ml, which is 40 times higher than the SN-38 titers recorded in patients treated with irinotecan.

[0195] We concluded that IMMU-132 is active (long-term stable disease) in 62% (8/13) of prostate cancer patients with multiple previous therapy failures, with manageable neutropenia and mild gastrointestinal (GI) toxicity. Patients with advanced prostate cancer may be prescribed repeated treatment cycles (more than 6) of 8-10 mg / kg IMMU-132 on days 1 and 8 of the 21-day cycle, with some dose adjustments or growth factor support for neutropenia in subsequent treatment cycles. These results are consistent with the results obtained in patients with advanced CRC, TNBC, SCLC, NSCLC, EAC, which demonstrated partial responses and long-term stable disease when IMMU-132 was administered. Thus, IMMU-132 monotherapy represents a new effective treatment regimen for patients with prostate cancer, including patients with tumors that were previously resistant to other treatment regimens for prostate cancer.

Methods and results

[0196] Expression of Trop-2 - Expression of Trop-2 on the surface of various cancer cell lines was determined by flow cytometry using QUANTBRITE® PE beads. For a number of Trop-2 molecules detected in different cell lines, the following results were obtained: Pancreatic cancer BxPC-3 (891000); stomach cancer NCI-N87 (383000); breast cancer MDA-MB-468 (341000); squamous cell lung cancer SK-MES-1 (27000); pancreatic cancer Capan-1 (115,000); stomach cancer AGS (78000) colon cancer COLO 205 (52000). Expression of Trop-2 was also observed in 29 of 29 (100%) tissue microarrays of pancreatic adenocarcinoma (not shown).

[0197] Accumulation of SN-38 - Accumulation of SN-38 was determined in nude mice with Capan-1 human pancreatic cancer xenografts (about 0.06-0.27 g). Mice were injected intravenously with irinotecan 40 mg / kg (773 μg; total SN-38 equivalents = 448 μg). This dose is the MTD (Maximum Tolerated Dose) for mice. Equivalent dose for humans = 3.25 mg / kg or about 126 mg / m ² . Or, mice were injected intravenously with IMMU-132 1.0 mg (SN-38: antibody ratio = 7.6; SN-38 equivalents = 20 μg). This dose is significantly lower than the maximum tolerated dose in mice. The equivalent human dose is about 4 mg / kg IMMU-132 (about 80 mcg / kg SN-38 equivalents). Autopsies were performed on 3 animals at a time interval, for mice that received irinotecan injections at time points of 5 min, 1, 2, 6 and 24 hours, or for mice that received injections of IMMU-132 at time points 1, 6, 24, 48 and 72 hours. Tissues were removed and analyzed by reverse phase HPLC analysis on SN-38, SN-38G and irinotecan. Extracts from animals treated with IMMU-132 were also acid hydrolyzed to release SN-38 from the conjugate (ie, SN-38 [TOTAL]). The results (not shown) demonstrate that IMMU-132 CAP has the ability to deliver 120 times more SN-38 to the tumor than irinotecan, although 22 times fewer SN-38 equivalents were injected with CAP.

[0198] Clinical Protocol IMMU-132 - The protocol used in the Phase I / II studies was as shown in Table 5 below.

Table 5. Clinical protocol using IMMU-132: OVERVIEW Dosing scheme Injection once a week for 2 weeks every 21 days for up to 8 cycles.
Patients with objective responses are allowed to continue treatment after 8 cycles. At initial enrollment, the administration was delayed and the planned dose was reduced at greater than or equal to the grade 2 toxicity associated with the treatment; the protocol was changed later in the course of the study so that delayed administration and dose reduction were applied only in cases of greater than or equal to grade 3 toxicity. The development of severe toxicity following treatment required dose reduction by 25% of the prescribed dose for the ^first case 1, 50% for 2 ^st case, and treatment was stopped completely at 3 ^meters case. Dose level cohorts 8, 12, 18 mg / kg; later reduced to an intermediate dose level of 10 mg / kg. Cohort size Phase I standard circuit [3 + 3]; the follow-up includes 15 patients for selected cancers. DLT (dose-limiting toxicity) ANC (absolute neutrophil count) grade 4 greater than or equal to 7 days; febrile neutropenia of any grade duration greater than or equal to 3; platelets grade 4 more than or equal to 5 days; hemoglobin grade 4; nausea / vomiting / diarrhea (N / V / D) grade 4 of any duration or any reaction of nausea / vomiting / diarrhea grade 3 for more than 48 hours; grade 3 infusion reactions; a non-heme toxicity of a grade greater than or equal to 3, at least likely due to the study drug. Maximum Allowable Dose (DMD) The maximum dose at which more than or equal to 2/6 of patients tolerate a full 21-day treatment cycle without delay or dose reduction or the development of toxicity is greater than or equal to 3 degrees. Patients • Metastatic colorectal, pancreas, stomach, esophagus, lung (NSCLC, SCLC), triple negative breast, prostate, ovary, kidney, bladder, head and neck, renal cell.
• Refractory / relapsing after standard treatment regimens for metastatic cancer.
• Prior irinotecan therapy is NOT required for enrollment.
• Absence of volumetric lesions more than 5 cm.
• At least 4 weeks after any "major" surgery, and 2 weeks after radiation or chemotherapy regimens.
• Gilbert's disease or known metastatic lesion of the central nervous system is excluded.

[0199] Patients were administered IMMU-132 in accordance with the above protocol. The following is a typical case report. A 34-year-old white male initially diagnosed with metastatic pancreatic (liver) cancer progressed on several chemotherapy regimens including gemcitabine / erlotinib / FG-3019, FOLFIRINOX and GTX prior to IMMU-132 ( dose 8 mg / kg on days 1 and 8 of the 21-day cycle). The patient received the drug for 4 months with good symptomatic tolerance, improvement in pain, a maximum decrease in CA19-9 by 72% (from 15885 U / ml to 4418 U / ml) and stable disease status according to RECIST-CT criteria along with evidence of tumor necrosis ... Therapy had to be suspended due to liver abscess; the patient died approximately 6 weeks later, 6 months after the initiation of therapy.

[0200] Studies 14 patients with advanced PCa who relapsed after an average of 2 previous therapies showed CT-confirmed antitumor activity, with 8/13 (62%) stable diseases. The median duration of URT for the 13 CT-evaluated patients was 12.7 weeks, compared with 8.0 weeks for the last prior therapy. This CAP, with a known nanomolar toxicity drug conjugated to an antibody targeting Trop-2, which is common in many epithelial cancers, using a linker that provides cleavage in the tumor focus, represents a new effective strategy in the therapy of pancreatic cancer with CAP. Compared to the current standard of care for patients with pancreatic cancer, the increase in time to progression in patients with pancreatic cancer, especially in patients resistant to multiple prior treatments, was unexpected and could not be predicted.

Example 5. Further results of phase I / II clinical trials

Triple negative breast cancer (TNBC)

[0201] The phase I / II clinical trials (NCT01631552) described in the Examples above were continued with 56 TNBC patients who received 10 mg / kg. The patient population had previously received intensive treatment prior to initiation of IMMU-132 therapy, with at least two previous lines of therapy, including taxane treatment. Previous treatments included cyclophosphamide, doxorubicin, carboplatin, gemcitabine, capecitabine, eribulin, cisplatin, anastrozole, vinorelbine, bevacizumab, and tamoxifen. Despite such an extensive medical history, patients with TNBC responded well to IMMU-132, with 2 confirmed complete responses (CR), 13 partial responses (PR) and 25 stable diseases (SD), with a total objective response of 29% (15/52 ) (Fig. 4). Summing up the rates of PO plus OR plus SD, TNBC treatment yielded 71% favorable responses for patients treated with IMMU-132 (not shown). The median time to progression in this previously intensively treated TNBC patient population was 9.4 months, ranging from 2.9 to 14.2 months to date. However, 72% of the patients in the study were still on treatment. Progression-free survival in this patient population is shown in FIG. 5.

Metastatic NSCLC

[0202] Clinical trials are also ongoing for patients with metastatic non-small cell lung cancer (NSCLC), with 29 evaluated patients enrolled to date who received 8 or 10 mg / kg IMMU-132. The best answers for RESIST 1.1 criteria are shown in FIG. 6. Of the 29 patients, 8 RRs and 13 SZs were observed. The time to progression for patients with NSCLC is shown in FIG. 7, which shows that 21/33 (64%) of patients with NSCLC showed OR or SD. The median time to progression was 9.4 (9/4) months, ranging from 1.8 to 15.5+ months, and 47% of patients were still on treatment. Progression-free survival in NSCLC patients treated with 8 or 10 mg / kg IMMU-132 is shown in FIG. 8. Median PFS was 3.4 months at 8 mg / kg and 3.8 months at 10 mg / kg. However, research is ongoing and mean progression-free survival is likely to improve.

Metastatic SCLC

[0203] Comparable results were obtained for patients with metastatic SCLC. The best RECIST 1.1 response for patients with metastatic SCLC who received 8 or 10 mg / kg IMMU-132 was 6 RR and 8 SD out of 25 patients evaluated (not shown). Time to progression showed a median of 4.9 months, ranging from 1.8 to 15.7+ months, and 7 patients continued to receive IMMU-132 treatment (not shown). Progression-free survival showed a median PFS of 2.0 months for 8 mg / kg and 3.6 months for 10 mg / kg (not shown). The median overall survival (OS) was 8.1 months for 8 mg / kg and could not yet be determined for 10 mg / kg.

[0204] Thus, ongoing phase I / II clinical trials show increased efficacy of IMMU-132 when administered with the indicated dosages of CAP, at least in TNBC, NSCLC, SCLC and urothelial cancers. The excellent therapeutic effect in these intensively treated and refractory metastatic cancers occurred without inducing severe toxicity that could preclude clinical use. IMMU-132 has demonstrated an acceptable safety profile in patients with various solid cancers previously treated with intensive therapy, on average, 2 -5 prior therapies. Neutropenia alone has shown an incidence of more than 20% of the patient population for grade 3 or higher adverse reactions. The study also demonstrates that repeated doses of IMMU-132 can be administered to humans at therapeutic doses without inducing host anti-IMMU-132 cross-reactive antibodies. These results demonstrate the safety and usefulness of IMMU-132 for the treatment of various Trop-2-positive cancers in humans.

Example 6. Combination therapy using CAP IMMU-132 and microtubule inhibitors or PARP inhibitors

[0205] Synthetic lethality is the concept in which a cell carrying one of two possible defects in a gene or protein is viable, whereas a cell containing both defects is not viable. BRCA1 / 2 mutations are associated with deficiencies in DNA repair and are associated with TNBC. Other repair mechanisms include poly (adenosine diphosphoribose) polymerase (PARP), which can be used by cancer cells to overcome BRACA1 / 2 loss. Treatment of TNBCs with IMMU-132 or paclitaxel leads to the degradation and deactivation of PARP, while olaparib, a small molecule, directly inhibits PARP. Thus, the hypothesis that combining IMMU-132 with paclitaxel or olaparib would effectively knock out PARP activity was investigated in TNBC xenografts to determine if such combinations would result in synthetic lethality.

[0206] The purpose of this study was to determine if the combination of an antibody-drug conjugate that induces DNA strand breaks, such as sacituzumab govitecan (also known as IMMU-132, anti-Trop-2 hRS7-CL2A-SN-38) , with microtubule inhibitors (eg paclitaxel or eribulin mesylate) or poly (adenosine diphosphoribose) polymerase (PARP) inhibitors (eg olaparib) in cancer (eg nude mice with TNBC xenografts) improve anti-tumor effects. One of ordinary skill in the art will understand that the unexpected improved effects of the combination of antibody-SN-38 conjugates with PARP or microtubule inhibitors are not limited to specific exemplary examples of an antibody, drug, PARP inhibitor, or microtubule inhibitor, but rather are specific to classes of antibodies against tumor associated antigens. (TAA), DNA strand break inducing drugs, PARP inhibitors, and microtubule inhibitors.

Experimental procedures

[0207] In a non-limiting example, mice with human TNBC (triple negative breast cancer) xenografts (MDA-MB-468 or HCC1806; about 0.3 cm ³ ) were treated with the maximum tolerated dose of paclitaxel (15 mg / kg weekly x 5 weeks) and IMMU-132 at 10 mg / kg or 12.5 mg / kg on days 1, 8, 22 and 29. Mice with HCC1806 tumors (about 0.28 cm ³ ) were treated with 2 cycles of IMMU-132 (12.5 mg / kg) and 0.5 mg / kg eribulin mesylate (equivalent to a human dose of 1.4 mg / m ² ) weekly for 2 weeks of a 21-day cycle. PARP inhibition studies used MDA-MB-468 tumor mice (about 0.32 cm ³ ) treated with olaparib (50 mg / kg, daily (qd) x 5 days x 4 weeks; 33% human dose equal 800 mg daily) and IMMU-132 (10 mg / kg, twice weekly x 4 weeks). Olaparib was administered as intraperitoneal (ip) injections daily for 5 consecutive days with two days of rest before repeating (qd × 5). This procedure was performed over a period of four weeks. IMMU-132 was injected intraperitoneally (ip) twice a week for four weeks. Control animals received non-tumor targeting anti-CD20 hA20-CL2A-SN-38 CAP, alone or in combination with olaparib. The primary endpoint was median survival time (MST), defined as the time to progression of tumors to 1.0 cm ³ .

[0208] In alternative embodiments, analysis of synergistic effects could be performed by in vitro analysis. Clonogenic analysis can be used to determine the viability of a cell fraction (Ibrahim et al., 2012, Cancer Discovery 2: 1036-47). Briefly, 350-800 cells are plated in 6-well flat-bottomed cell culture plates in two parallel runs. Twenty-four hours after seeding, cells are washed and fresh medium is added in the presence of increasing doses of CAP and / or PARP or a microtubule inhibitor (eg, olaparib), alone or in combinations. Media containing drug (and / or) is renewed on day 4. Colonies are fixed and stained after 7 days of treatment with 1.5 ml of 6.0% glutaraldehyde and 0.5% crystal violet, and colonies are counted according to standard procedures. The surviving cell fraction (SF) is calculated as follows:

SF =

where Sowing efficiency factor =

[0209] The interaction between CAP and PARP or microtubule inhibitor is assessed using the Chou and Talalay multiple drug effects assay (1984, Adv Enzyme Regul 22: 27-55). This method quantifies interactions between two or more drugs, with values less than 1 indicating synergistic interactions, values greater than 1 indicating antagonistic interactions, and values equal to 1 indicating additive interactions.

results

[0210] Mice with MDA-MB-468 tumors treated with the combination of IMMU-132 and paclitaxel showed superior antitumor effects (not shown), with more than 11-fold reduction in tumor versus 1.4-fold reduction in the IMMU single group −132 (P = 0.0003; area under the curve, AUC) or 11.4-fold increase in tumor size in mice treated with paclitaxel alone (P <0.0001; AUC).

[0211] For MDA-MB-468, the combination of 200 μg IMMU-132 plus paclitaxel gave an excellent antitumor effect in terms of area under the curve (AUC) compared to all other groups (Table 6, P less than 0.0013). Reducing the amount of IMMU-132 administered with paclitaxel to 100 μg also resulted in significant antitumor effects compared to mice treated with paclitaxel alone, only IMMU-132 (100 μg), or untreated animals (Table 7, P less than 0 , 0328). Further comparisons of growth curves for paclitaxel groups or untreated control groups cannot be made as each of them began to lose mice due to disease progression (i.e., tumor volume (TV) greater than 1.0 cm ³ ), as of day 49 of therapy.

Table 6. Area under curves comparison between MDA-MB-468 tumor carrier mice treated with IMMU-132 (200 μg) plus paclitaxel and all other treatment groups. Treatment Comparison time Tumor volume (cm ³ )
on that day (mean ± standard deviation) P-value
(AUC) IMMU-132 (200 mcg)
a plus
paclitaxel
against (vs.) IMMU-132 (200 mcg) until day 98 of therapy 0.162 ± 0.144 vs. 0.621 ± 0.324 0.0003 IMMU-132 (100 mcg) until day 70 of therapy 0.050 ± 0.062 vs. 0.634 ± 0.335 0.0002 paclitaxel until day 49 of therapy 0.025 ± 0.041 vs. 0.705 ± 0.206 less than 0.0001 IMMU-132 (100 mcg) + paclitaxel until day 112 of therapy 0.202 ± 0.191 vs. 0.496 ± 0.286 0.0013 without treatment until day 49 of therapy 0.025 ± 0.041 vs. 0.663 ± 0.349 less than 0.0001

Table 7. Area under curves comparison between MDA-MB-468 tumor carrier mice treated with IMMU-132 (100 μg) plus paclitaxel and all other treatment groups. Treatment Comparison time Tumor volume (cm ³ )
on that day (mean ± standard deviation) P-value
(AUC) IMMU-132 (100 mcg)
a plus
paclitaxel
against (vs.) IMMU-132 (200 mcg) until day 98 of therapy 0.663 ± 0.349 vs. 0.621 ± 0.324 0.9539 IMMU-132 (100 mcg) until day 70 of therapy 0.311 ± 0.196 vs. 0.634 ± 0.335 0.0328 paclitaxel until day 49 of therapy 0.211 ± 0.155 vs. 0.705 ± 0.206 less than 0.0001 without treatment until day 49 of therapy 0.211 ± 0.155 vs. 0.663 ± 0.349 0.0001

[0212] For rapidly progressing HCC1806 xenografts (Figs. 9A-9B), the combination of IMMU-132 plus paclitaxel showed an excellent antitumor effect compared to IMMU-132 monotherapy (P = 0.0195, AUC _{17 days} ). It is a very aggressive tumor with a median survival time (MVT) of only 10 days after initiation of therapy for untreated control animals (18 days after tumor cell inoculation). In terms of survival, the combination that achieved an MVI of 38 days provided a significant improvement in survival compared to all other therapies (P <0.017; log-rank). It should be noted that this result was achieved at a low dose of only 0.25 mg, which would be equivalent to a human dose of only 1 mg / kg.

[0213] Mice treated with the combination of IMMU-132 plus eribulin mesylate (not shown) showed a significantly higher antitumor response than all other monotherapy groups (P less than 0.0432; two-tailed t-test). This resulted in a significant increase in survival for the combination (MVI = 23 days) compared with eribulin or IMMU-132 monotherapy (MVV = 18 and 14 days, respectively; P <0.0044; log-rank).

[0214] Similarly, the combination of IMMU-132 therapy with olaparib was superior to single agent therapy in mice with MDA-MB-468 tumors (P less than 0.0032; AUC). The results are summarized in Table 8. All IMMU-132 combination therapies were well tolerated.

Table 8. Area under curves comparison between MDA-MB-468 tumor carrier mice treated with IMMU-132 plus olaparib and all other treatment groups. Treatment Comparison time Tumor volume (cm ³ )
on that day (mean ± standard deviation) P-value
(AUC) IMMU-132
a plus
olaparib
against (vs.) only IMMU-132 until day 49 of therapy 0.030 ± 0.038 vs. 0.088 ± 0.069 0.0023 hA20-SN-38 plus olaparib until day 49 of therapy 0.030 ± 0.038 vs. 0.652 ± 0.306 less than 0.0001 only hA20-SN-38 until day 42 of therapy 0.045 ± 0.045 vs. 0.654 ± 0.285 0.0002 only olaparib until day 28 of therapy 0.083 ± 0.050 vs. 0.649 ± 0.267 less than 0.0001 saline until day 28 of therapy 0.083 ± 0.050 vs. 0.697 ± 0.352 less than 0.0001

[0215] Determination of drug-antibody ratio (DAR). Five clinical lots of IMMU-132 were evaluated by hydrophobic interaction HPLC (HIC-HPLC), which identified three resolved peaks corresponding to products with DAR 6, 7 and 8, with the largest fraction containing the product with DAR = 8 (not shown). This method provides stable production of IMMU-132 with a total DAR (DAR _AVE ) of 7.60 ± 0.03 for five clinical batches. HIC-HPLC results were confirmed by liquid chromatography-mass spectrometry (LC-MS). Analysis showed that over 99% of the 8 available sulfhydryl groups were coupled with the CL2A linker, with or without SN-38 attached (not shown). No unsubstituted (or terminal N-ethylmaleimide groups) heavy or light chains were found. Thus, the differences in DAR between the products are caused by the release of SN-38 by the linker during preparation, and not by the lower initial degree of substitution. After receiving and lyophilization, IMMU-132 remained stable for several years.

[0216] The effect of DAR on pharmacokinetics and antitumor efficacy in mice. Mice with Trop-2 ⁺ human gastric cancer xenografts (NCI-N87) received 2 courses of treatment, 7 days apart, each with equal doses of IMMU-132 protein (0.5 mg) having DAR values of 6.89, 3. 28 or 1.64. Animals treated with PAP with DAR 6.89 had significantly improved median survival time (MVT) compared to mice treated with PAP with DAR 3.38 or 1.64 (MVI = 39 days vs. 25 and 21 days, respectively; P less than 0.0014) (not shown). There was no difference between the groups receiving conjugates with DAR 3.28 or 1.64 and the saline control group.

[0217] To further clarify the importance of higher DAR values, mice with gastric tumors NCI-N87 were injected with 0.5 mg of IMMU-132 with DAR 6.89 twice a week for two weeks (not shown). Another group received twice the protein dose (1 mg) of IMMU-132 conjugate with DAR 3.28. Although both groups received the same total amount of SN-38 (36 μg) in each dosing regimen, the group receiving the DAR 6.89 conjugate inhibited tumor growth to a much greater extent than the tumor carrier animals receiving the DAR conjugate 3.28 (P = 0.0227; AUC) (not shown). In addition, treatment with a lower DAR product did not significantly differ from untreated controls. Taken together, these studies show that a lower DAR value decreases efficiency.

[0218] A study of the pharmacokinetic behavior of conjugates prepared with these different ratios was performed in tumor-free mice treated with 0.2 mg of each conjugate, unconjugated hRS7 IgG, or hRS7 IgG reconstituted followed by terminal N-ethyl maleimide groups. 5 serum samples were taken at time points from 0.5 to 168 h and analyzed by ELISA for hRS7 IgG. There was no significant difference in clearance of these conjugates compared to unconjugated IgG (not shown). Thus, the level of substitution did not affect the pharmacokinetics of the conjugates and, no less important, the restoration of interchain disulfide bonds, apparently, did not destabilize the antibody.

[0219] The mechanism of action of IMMU-132 in TNBC. The apoptotic pathway used by IMMU-132 was investigated in the TNBC cell line, MDA-MB-468, and in the HER2 ⁺ SK-BR-3 cell line, to confirm that the functioning of the CAP is based on its constituent SN-38. Cells were exposed to 1 μM SN-38, SN-38 equivalent of IMMU-132, or protein equivalent of hRS7. Cells were harvested and Western blotted. SN-38 and IMMU-132 taken separately mediated a more than 2-fold increase in p21 ^{WAF1 / Cip1} within 24 h in MDA-MB-468, and after 48 h the amount of p21 ^{WAF1 / Cip1} in these cells began to decrease (31% and 43% for SN-38 or IMMU-132, respectively) (not shown). Interestingly, in the HER2 ⁺ SK-BR-3 tumor line, neither SN-38 nor IMMU-132 mediated an increase in ^{WAF1 / Cip1 p21} above constitutive levels in the first 24 hours, but, as seen for MDA-MB-468 cells after 48 h exposure to SN-38 or IMMU-132, the amount of p21 ^{WAF1 / Cip1} decreased by more than 57% (not shown). Both SN-38 and IMMU-132 led to the cleavage of procaspase-3 into its active fragments within 24 hours, but with a greater degree of active fragments observed after exposure for 48 hours. It should be noted that in both cell lines, IMMU-132 mediated a higher degree of pro-caspase-3 degradation, with the highest level observed after 48 hours compared to cells exposed to SN-38 (not shown). Finally, SN-38 and IMMU-132 both mediated the cleavage of poly (ADP-ribose) polymerase (PARP) starting at 24 hours, with almost complete cleavage at 48 hours (not shown). Taken together, these results confirm that IMMU-132 has a mechanism of action similar to free SN-38 when administered in vitro.

[0220] Comparison of SN-38 delivery by IMMU-132 and irinotecan in a human tumor xenograft model. Constitutive products derived from irinotecan or IMMU-132 were determined in serum and tumors of mice implanted with a subcutaneous (sc) human pancreatic cancer xenograft (Capan-1) injected with irinotecan (773 μg; SN-38 equivalents = 448 μg) and IMMU-132 (1.0 mg; SN-38 equivalents = 16 μg). At 5 intervals after administration, 3 animals from each group were euthanized and serum extracted to isolate products of interest.

[0221] Irinotecan is very rapidly cleared from serum, with conversion to SN-38 and SN-38G observed within 5 minutes (not shown). None of the products were detected after 24 hours.The AUC (area under the curve) values over a time period of 6 hours were 21.0, 2.5, and 2.8 μg / ml · h for irinotecan, SN-38, and SN -38G, respectively (SN-38 conversion in mice = [2.5 + 2.8) / 21 = 25.2%]). Animals treated with IMMU-132 had much lower serum concentrations of free SN-38, but was detectable within 48 hours (not shown). Free SN-38G was detected only at 1 and 6 hours, and had levels 3-7 times lower than free SN-38 (not shown).

[0222] In Capan-1 tumors excised from irinotecan-treated animals, irinotecan levels remained high for 6 hours, but were not detected after 24 hours (AUC _5min-6h = 48.4 μg / g · h). SN-38 levels were significantly lower and were only detectable for 2 h (i.e., AUC _{5 min-} 2 h = 0.4 μg / g h), and SN-38G values were almost 3 times higher (AUC = 1.1 μg / g h) (not shown). Tumors taken from animals treated with IMMU-132 did not contain detectable free SN-38 or SN-38G, and all SN-38 in the tumor was associated with IMMU-132. It is important to note that since SN-38G was not detected in tumors, it can be assumed that the SN-38 associated with IMMU-132 was not glucuronidated. The AUC value for IMMU-132 associated SN-38 in these tumors was 54.3 μg / g h, which is 135 times higher than the amount of SN-38 in tumors of animals treated with irinotecan during the 2-hour period when SN-38 could be detected despite the fact that mice treated with irinotecan received 28 times more SN-38 equivalents than when administered with IMMU-132 (i.e., 448 and 16 μg SN-38 equivalents, respectively)

conclusions

[0223] IMMU-132 is a humanized anti-Trop-2 antibody conjugated to 7.6 molecules of SN-38, an active metabolite of irinotecan, an inhibitor of topoisomerase I. IMMU-132 has clinically demonstrated controlled toxicity and encouraging responses in patients with relapsed / refractory TNBC (ClinicalTrials.gov, NCT01631552). The IMMU-132 therapy alone demonstrated significant antitumor effects in xenografts of human TNBC at an equivalent human dose, 5 times less than in clinical use (i.e. 10 mg / kg). Since preclinical studies show that IMMU-132 can be combined with two different microtubule inhibitors or PARP inhibitor with significantly increased antitumor activity, these data support the use of IMMU-132 and other DNA-breakable antibody drug conjugates (ADCs) in combination with microtubule inhibitors and / or PARP inhibitors in general, as well as other chemotherapeutic agents that target cell division by inhibiting microtubules or repairing DNA. The preferred class of CAP is anti-Trop-2 antibody conjugates for patients with Trop2-positive cancers, including but not limited to TNBC, metastatic colon cancer, SCLC, NSCLC, and urothelial cancer, as it is a target that is expressed in high amounts at high numbers. cancer and is localized on the cell surface and cytoplasmically in cancer cells.

[0224] Synergy was achieved when IMMU-132 was combined with PARP inhibitors (eg, olaparib) in TNBCA tumor lines having BRCA1 / 2 defects, as well as wild-type expression, including only PTEN-deficient. This suggests that IMMU-132 can synergistically interact with any tumor that has any disruption in the homologous DNA recombination pathways. In combination with olaparib, IMMU-132 therapy achieved significant anti-tumor effects, exceeding those observed with each of them alone, which led to a significant increase in survival. IMMU-132 in combination with microtubule inhibitors (such as paclitaxel or eribulin mesylate) also significantly increased efficacy compared to monotherapy with each agent.

[0225] Overall, these data indicate an unexpected significant advantage of combination therapy with an antibody-drug conjugate (CAP), which targets cancer cells and induces DNA strand breaks, such as IMMU-132, and microtubule inhibitors or PARP inhibitors. Targeting the PARP DNA repair pathway in BRCA1 / 2 mutant TNBC tumors by combining IMMU-132 therapy with paclitaxel or olaparib achieved synthetic lethality in this disease model with no apparent toxicity. In a typical implementation, the combination of IMMU-132 and a PARP or microtubule inhibitor is used to treat Trop-2 positive cancers such as urothelial cancer. These data support the use of IMMU-132 in combination with other chemotherapeutic agents that also target DNA repair mechanisms in patients with urothelial or similar tumors.

[0226] From the above description, a person skilled in the art can easily determine the main characteristics of this invention and, without going beyond its essence and scope, can make various changes and modifications of the invention to adapt it to different applications and conditions without undue experimentation. All patents, patent applications and publications mentioned in this document are incorporated by reference.

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LIST OF SEQUENCES

<110> IMMUNOMEDICS, INC.

<120> METASTATIC UROTHELIAL CANCER THERAPY USING

ANTIBODY-DRUG CONJUGATE SACITUZUMAB GOVITEKAN

(IMMU-132)

<130> IMM356WO2

<140>

<141>

<150> 62 / 428.655

<151> 2016-12-01

<160> 9

<170> PatentIn version 3.5

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Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

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Claims (18)

1. A method for the treatment of metastatic urothelial cancer, comprising administering to a human patient with urothelial cancer an antibody-drug conjugate (CAP) containing SN-38 conjugated to an anti-Trop-2 antibody hRS7 or its antigen-binding fragment, said CAP being administered in a dose 8 mg / kg or 10 mg / kg, wherein said patient has relapsed after one or more prior standard therapy and checkpoint inhibitor therapy, or was refractory to one or more previous standard therapy and therapy with checkpoint inhibitors before treatment with said CAP, where said CAP has structure MAT-CL2A-SN-38

2. The method of claim 1, wherein the one or more prior conventional therapy comprises platinum-containing chemotherapy. 3. The method of claim 1, wherein the checkpoint inhibitor therapy comprises an anti-PD-1 antibody or an anti-PD-L1 antibody. 4. The method according to claim 1, wherein at least 6 SN-38 molecules are attached to each antibody molecule. 5. The method according to claim 1, characterized in that 6-8 SN-38 molecules are attached to each antibody molecule. 6. The method according to claim 1, characterized in that the dose of said CAP is administered to a human patient once a week on a schedule with a cycle of two weeks of therapy followed by one week off. 7. The method according to claim 6, characterized in that the cycle is repeated 4, 6, 8, 10, 12, 16 or 20 times. 8. The method according to claim 1, characterized in that said CAP is administered in combination with one or more therapeutic agents selected from the group consisting of an antibody, an antigen-binding fragment of an antibody, a drug, a toxin, an enzyme, a hormone, immunomodulators, an antisense oligonucleotide, photoactive agent, radioisotope, PARP inhibitor, microtubule inhibitor, Bruton's kinase inhibitor and PI3K inhibitor. 9. The method according to claim 8, characterized in that said drug or toxin is selected from the group consisting of 5-fluorouracil, afatinib, aplidine, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin , bortezomib, bosutinib, bryostatin-1, busulfan, calicheamicin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustin, celecoxib, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (SN-38, 11), cladribine kamptotekanov, cyclophosphamide, krizotiniba, cytarabine, dacarbazine, dasatinib, dinatsikliba, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolino (2P-DOX), tsianomorfolinodoksorubitsina, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epipodophyllotoxin, erlotinib, entinostata , estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, flavopiridol, floxuridine (FUdR ), 3 ', 5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl protein transferase inhibitors, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine and hydroxyruburea, idealisib, ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, neocarcinylamide olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustin, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide, transplatin, thalidomide, thiothepidoguanine, tenipotin vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839. 10. The method according to claim 8, wherein the PARP inhibitor is selected from the group consisting of olaparib, thalazoparib (BMN-673), rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699, BSI -201, CEP-8983 and 3-aminobenzamide. 11. The method of claim 8, wherein the PARP inhibitor is olaparib. 12. The method according to claim 8, wherein the microtubule inhibitor is selected from the group consisting of vinca alkaloid, taxane, maytansinoid, auristatin, vincristine, vinblastine, paclitaxel, mertansine, demecolcine, nocodazole, epothilone, docetolide discoderm, discoderm colchicine, combrestatin, podophyllotoxin, CI-980, phenylagistins, steganacins, curacins, 2-methoxyestradiol, E7010, methoxybenzenesulfonamides, vinorelbine, vinflunine, vindesine, dolastatins, spongystatin, tsidinastinetrietinov, EGM and eribulin mesylate. 13. The method of claim 8, wherein the microtubule inhibitor is paclitaxel or eribulin mesylate. 14. The method according to claim 8, wherein the Bruton kinase inhibitor is selected from the group consisting of ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059, GDC-0834, LFM -A13 and RN486. 15. The method of claim 8, wherein the Bruton's kinase inhibitor is ibrutinib. 16. The method according to claim 8, wherein the PI3K inhibitor is selected from the group consisting of idealisib, wortmannin, demethoxyviridine, perifosin, PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, palomida 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZ4002. And LY29 17. The method of claim 8, wherein the PI3K inhibitor is idealisib.

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