ES2886927T3 - Immunoconjugates of antibody-SN-38 with a CL2A linker - Google Patents

Abstract

A method for producing a compound, CL2A- SN-38, of the structure, **(See formula)** comprising carrying out a reaction scheme as shown: **(See formula)** wherein intermediate 9 does not is exposed to triphenylphosphine.

Description

DESCRIPTION

Immunoconjugates of antibody-SN-38 with a CL2A linker

FIELD OF THE INVENTION

[0001] The present invention relates to a method for producing the compound CL2A-SN-38. Making use of such a method, an improved synthetic protocol for producing therapeutic immunoconjugates that can target cancer cells, infectious disease organisms, and/or cells associated with autoimmune diseases is provided. The antibody and drug moieties of said therapeutic immunoconjugate are joined by an intracellularly cleavable bond that increases therapeutic efficacy. The new synthetic protocol provides improved efficiency of synthesis of CL2A-SN-38 conjugates, with substantially lower levels of contaminating by-products.

BACKGROUND OF THE INVENTION

[0002] For many years it has been a goal of scientists in the field of targeted drug therapy specifically to use monoclonal antibodies (MAbs) for the specific delivery of toxic agents to human cancers. Suitable tumor-associated MAb conjugates and toxicants have been developed, but have had mixed success in cancer therapy and virtually no application in other diseases, such as infectious and autoimmune diseases. The toxic agent is most commonly a chemotherapeutic drug, although particle-emitting radionuclides or bacterial or plant toxins have also been conjugated to MAbs, especially for cancer therapy (Sharkey and Goldenberg, CA Cancer J Clin. 2006 Jul-Aug; 56 ( 4):226-243) and, more recently, with radioimmunoconjugates for preclinical therapy of certain infectious diseases (Dadachova and Casadevall, QJ Nucl Med Mol Imaging 2006;50(3):193-204).

[0003] The advantages of using MAb-chemotherapeutic drug conjugates are that (a) the chemotherapeutic drug itself is structurally well defined; (b) the chemotherapeutic drug is bound to the MAb protein using well-defined conjugation chemistries, often at specific sites remote from the MAb antigen-binding regions; (c) MAb-chemotherapeutic drug conjugates can be prepared more reproducibly than chemical conjugates involving MAbs and bacterial or plant toxins, and as such are more amenable to commercial development and regulatory approval; and (d) Mab-chemotherapeutic drug conjugates are orders of magnitude less systemically toxic than MAb radionuclide conjugates.

[0004] Early work on protein-drug conjugates indicated that a drug is preferably released in its original form, once it has been internalized into a target cell, for the protein-drug conjugate to be a useful treatment. Trout et al. (Proc. Natl. Acad. Sci. USA 79: 626-629 (1982)) showed the advantage of using specific peptide linkers, between the drug and the rest of the antibody, that lysosomally cleave to release the drug intact. In particular, MAb-chemotherapeutic drug conjugates prepared using mild acid-cleaving linkers, such as those containing a hydrazone, were developed based on the observation that the pH within tumors was often lower than normal physiological pH ( Willner et al., US Patent 5,708,146; Trail et al. (Science 261:212-215 (1993)). The first approved MAb-drug conjugate, gemtuzumab ozogamicin, incorporated an acid-labile hydrazone bond between a anti-CD33 antibody, humanized P67.6 and a potent calicheamicin derivative Sievers et al J Clin Oncol 19: 3244-3254 (2001) Hamann et al Bioconjugate Chem 13: 47-58 (2002) In some cases, MAb-chemotherapeutic drug conjugates were prepared with reductively labile hindered disulfide bonds between the chemotherapeutic drug and the MAb (Liu et al, Proc Natl Acad Sci USA 93: 8618-8623 (1996)).

[0005] Yet another cleavable linker involves cathepsin B-labile dipeptide spacers, such as Phe-Lys or Val-Cit, similar to the lysosomally labile peptide spacers of Trouet et al. containing one to four amino acids, further incorporating a collapsible spacer between the drug and the dipeptide (Dubowchik, et al., Bioconjugate Chem. 13:855-869 (2002); Firestone et al., US Pat. 6,214,345 B1; Doronina et al., Nat Biotechnol. 21: 778-784 (2003 )). The latter approaches were also used in the preparation of a camptothecin immunoconjugate (Walker et al., Bioorg Med Chem Lett. 12:217-219 (2002)). Another cleavable moiety that has been explored is an ester bond incorporated in the linker between the antibody and the chemotherapeutic drug. Gillimard and Saragovi have discovered that when an ester of paclitaxel is conjugated with p75 anti-rat MAb, MC192, or anti-human TrkA MAb, 5C3, the conjugate was found to exhibit target-specific toxicity. Gillimard and Saragovi, Cancer Res. 61: 694-699 (2001).

[0006] Current notions of antibody-drug conjugate design emphasize the use of ultratoxic drugs attached to antibodies using stable linkages that are cleaved only intracellularly. This approach has been used to design ultratoxic drug conjugates, such as calicheamicin, monomethylauristatin-E (MMAE), and maytansinoids. Although very stable binding to MAbs results in stability in the circulation, the conjugates are also processed in the liver, spleen, and kidney, thereby releasing toxic drugs in those organs and potentially narrowing the therapeutic window in treatment applications. of diseases. Although the recent regulatory approval of ADCETRIS® (brentuximab vedotin) for Hodgkin's lymphoma and KADCYLA® (adotrastuzumab emtansine) for refractory breast cancer is encouraging, the lack of therapeutic efficacy at the maximum administrable dose of calicheamicin conjugate in non-Hodgkin's lymphoma, and its subsequent discontinuation, as well as the withdrawal from the market of gemtuzumab ozogamicin for acute myeloid leukemia, note the Limitations of the use of ultratoxic agents in ADCs.

[0007] The conjugates of the present invention possess greater efficacy than unconjugated or "naked" antibodies or antibody fragments, although such unconjugated antibody moieties have been of use in specific situations. In cancer , eg. For example, naked antibodies have come to play a role in the treatment of lymphomas (alemtuzumab and rituximab), colorectal and other cancers (cetuximab and bevacizumab), breast cancer (trastuzumab), as well as a large number now in clinical development ( eg, epratuzumab, veltuzumab, milatuzumab). In most of these cases, clinical use has involved combining these naked or unconjugated antibodies with other therapies, such as chemotherapy or radiation therapy.

[0008] The use of CL2A linkers to attach therapeutic drugs, such as SN-38, to antibody moieties has been described (eg, US Patent Nos. 7,999,083 and 8,080,250). However, there is a need for more efficient methods to prepare and use CL2A and MAb-CL2A-SN-38 conjugates, including optimized dosing schedules that result in maximal efficacy and minimal toxicity, as well as efficient large-scale production. of CL2A-SN-38. and antibody-CL2A-SN-38 conjugates.

[0009] US 2014/0227180 A1 relates to methods and compositions for preparing SN-38 conjugates of proteins or peptides.

[0010] US 2014/0170063 A1 relates to therapeutic immunoconjugates comprising SN-38 linked to an antigen-binding antibody or antibody fragment.

[0011] US 2011/0305631 A1 relates to compositions and methods of use comprising combinations of anti-CD22 antibodies with a therapeutic agent.

[0012] US 7,999,083 B2 relates to therapeutic conjugates with improved ability to target various diseased cells containing a targeting moiety such as an antibody or antibody fragment, a linker and a therapeutic moiety, and processes for making and use of conjugates.

SUMMARY OF THE INVENTION

[0013] The problem underlying the present invention is solved by the subject matter of the appended independent claim; preferred embodiments can be taken from the attached dependent claims.

[0014] More specifically, the problem underlying the present invention is solved in a first aspect by a process for producing a compound, CL2A-SN-38, of the structure,

which comprises carrying out a reaction scheme as shown:

^{(I) PABOH e e or q , CH.Cl,}

Fmoc- lvs(m MT>OH LysiMMT)-PABOH

CL2A-SN-38

FIG. one

where intermediate 9 is not exposed to triphenylphosphine.

[0015] In an embodiment of the first aspect, the method further comprises using a 1.1-fold molar excess of tetrabutylammonium fluoride to remove a silyl protecting group and convert intermediate 6 to intermediate 7.

[0016] In an embodiment of the first aspect, the method further comprises performing three washes of an organic extract comprising intermediate 6 with 0.05 M sodium acetate buffer, pH 5.3.

[0017] In an embodiment of the first aspect, the method further comprises performing two washes of an organic extract comprising intermediate 7 with 0.25 M sodium citrate buffer, pH 6.

[0018] In an embodiment of the first aspect, the method further comprises purifying intermediate 7 by stepwise elution from a silica gel column with a mixture of dichloromethane, ethyl acetate and methanol.

[0019] In an embodiment of the first aspect, the method further comprises

a) forming a mixture comprising biphasic, (i) copper sulfate and sodium ascorbate in water; and (ii) intermediate 7, intermediate 8 and 2,6-collidine in dichloromethane; Y

b) stirring the biphasic mixture overnight at room temperature.

[0020] In an embodiment of the first aspect, the method further comprises

a) purification of intermediate 9 by EDTA washing and chromatography; Y

b) concentration of intermediate 9 to give a solid for storage, before removal of a silyl group from intermediate 9 to form CL2A-SN-38.

[0021] In an embodiment of the first aspect, the method further comprises (i) forming a solution of solid intermediate 9; and (ii) reacting intermediate 9 in solution with dichloroacetic acid and anisole to form CL2A-SN-38.

[0022] In an embodiment of the first aspect, the method further comprises precipitating CL2A-SN-38 by dropwise addition of tert-butyl methyl ether (t-BME).

[0023] In a second aspect, the problem underlying the present invention is solved by a method for the preparation of an SN-38 conjugated protein or peptide comprising the method of the first aspect, including any embodiment thereof, wherein the method further comprises reacting a maleimide moiety of CL2A-SN38 with a reduced sulfhydryl in a protein or peptide to produce an SN38-conjugated protein or peptide.

[0024] In an embodiment of the second aspect, the protein or peptide is an antigen-binding antibody or antibody fragment and the SN-38 conjugated antibody or antibody fragment is an immunoconjugate.

[0025] In an embodiment of the second aspect, the method further comprises purifying the immunoconjugate by tangential flow filtration (TFF), wherein the TFF is performed with a 50,000 dalton molecular weight membrane cut-off using volumes of 25 at 30 buffer diafiltration.

[0026] In an embodiment of the second aspect, the method further comprises formulating the immunoconjugate in Good's biological buffer at a pH of 6.0 to 7.0, and lyophilizing the immunoconjugate for storage, wherein the Good's biological buffer Good is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 1,4-piperazinediethanesulfonic acid (TUBOS), in the pH range of 6-7, preferably in the pH range of 6.5 to 7, and in a buffer concentration of 10-100 mM.

[0027] In one embodiment, the antibody or antibody fragment is linked to between 6 and 8 copies of CL2A-SN38.

[0028] In an embodiment of the second aspect, the antibody is selected from the group consisting of LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (anti-CD22), RS7 (anti-EGP-1) , PAM4 (anti-MUC5AC), KC4 (anti-mucin), A19 (anti-CD19), A20 (anti-CD20), MN-14 (anti-CEACAM5), MN-15 (anti-CEACAM6), MN-3 (anti-CEACAM6), R1 (anti-IGF-1R), Mu-9 (antiCSAp), Immu 31 (anti-AFP), CC49 (anti-TAG-72), J591 (anti-PSMA), HuJ591 (anti- PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX), and hL243 (anti-HLA-DR).

[0029] The present invention as defined in the claims addresses an unmet need in the art by providing improved methods for preparing SN-38-antibody immunoconjugates. The methods described are useful for the treatment of a variety of diseases and conditions that are refractory or less responsive to other forms of therapy, and may include diseases against which suitable antibodies or antigen-binding antibody fragments can be developed for the selective addressing, are either available or known. Preferred diseases or conditions that can be treated with the immunoconjugates prepared according to the present invention as defined in the claims include, e.g. eg, cancer, autoimmune disease, immune dysfunction disease, or diseases caused by infectious organisms.

[0030] The invention as defined in the claims relates to a novel synthetic protocol for the production of CL2A-SN-38 and mAb-Cl2A-SN-38 conjugates, as exemplified in FIG. 1. Unexpectedly, the new synthetic protocol shows improved production efficiency and yield, with substantially reduced levels of contaminating by-products. In specific embodiments, the new synthetic protocol may include the use of particular intermediates, precursors, reaction steps, and/or reaction conditions that are considered advantageous in the practice of the present invention.

[0031] Specific steps or conditions that have been found to be unexpectedly advantageous in the practice of the invention as defined in the claims may include one or more of the following. (i) In the conversion of silyl-protected intermediate 6 (Figure 1) to desilylated intermediate 7 (Figure 1), tetrabutylammonium fluoride was used in a 1.1-fold molar excess, instead of a 1-fold molar excess, 4 times as described above. (eg, US Patent No. 9,107,960). This level of reagent was equally effective in desilylation, while reducing the impurity profile, thus improving the purity of Intermediate 7 and leading to improved yield. (ii) In the aqueous treatments in the preparation of both intermediates 6 and 7 (Figure 1), as previously described (US Patent No. 9,107,960), the treatment involved washing the organic extracts four times with buffer of 0.05 M sodium acetate, pH5.3 for intermediate 6 and four times with 0.25 M citrate buffer, pH 6 for intermediate 7, with washings with water subsequently in each case. In an improved run, the buffer washes were reduced to 3 times for Intermediate 6 and 2 times for Intermediate 7, and the water washes were eliminated in both cases. These modifications considerably reduced the formation of aqueous-organic emulsion and improved the recovery of the product in the organic extract. (iii) Chromatographic purification of intermediate 7 (Figure 1) described in US Patent No. 9,107,960 involved the use of dichloromethane-methanol mixtures for elution. In an improved process, the elution was modified to use a combination of dichloromethane-ethyl acetate-methanol mixtures with varying concentration of methanol, followed by varying concentration of dichloromethane-methanol mixtures. This resulted in better and faster separation of the intermediate and improved recovery. Using these improved procedures, the overall yield in the 3-step sequence from Intermediate 4 to Intermediate 7 was improved from a range of 29-40% to 64% by incorporating these process changes.

[0032] The synthetic protocol for CL2A-SN-38 may further utilize one or more of the following enhancements. (iv) In a previously reported synthetic protocol (US Patent No. 9,107,960), a copper (+1) catalyzed reaction was performed between intermediates 7 and 8 (Figure 1) to produce intermediate 9 (Figure 1 ) using a complex formed of cuprous bromide and triphenylphosphine in dichloromethane with the addition of a tertiary amine, such as diisopropylethylamine. It was later discovered that in the presence of triphenylphosphine alone, the quality of the product of intermediate 7 deteriorated over time, indicating a deleterious effect of this reagent, which required its rapid removal once the copper-catalyzed reaction was complete. This has the potential to reduce the yield of purified intermediate 9 (Figure 1). As described in the examples below, in the present protocol the reaction was performed using a biphasic mixture containing copper sulfate and ascorbic acid in water and intermediates 7 and 8, as well as 2,6-collidine, in dichloromethane. By stirring the biphasic solution overnight, typically 18 h, the conversion was complete and the crude product was separated from copper sulfate and ascorbate by a simple wash with aqueous EDTA. This was followed by chromatographic purification, which led to a better yield of the product. The modified procedure avoids the use of triphenylphosphine. (v) The stability profile of intermediate 9 (Figure 1) in solution and as a solvent-free isolate showed that the product maintained >97% purity when stored as a solid product at -20°C for 4 days or as a solution in dichloromethane at room temperature for 1 day. In the latter case, storage at room temperature for 4 days reduced the purity to 89.4%, while the solution stored at -20°C showed only 92% purity. As previously described in US Patent No. 9,107,960, the material in dichloromethane, obtained after chromatography and EDTA washing, was used as is for the dichloroacetic acid (DCA) and anisole mediated deprotection reaction. In that situation, depending on the duration of storage of intermediate 9 in solution form, the quality of the final material (product 10, which is CL2A-SN-38) varied. In an improved process, intermediate 9 (Figure 1) after purification was concentrated to a solid which was used later in the final step. This modification removed uncertainty about changes in product quality of Intermediate 9 before the final deprotection step. An additional change in this step involved adding the crude product solution, after reaction, dropwise over several hours, into vigorously stirred tert-butyl methyl ether (t-BME), which allowed precipitation of the product as filterable solid. If the addition of the reaction mixture is not gradual and if the stirring is not vigorous, the product is oily rather than solid. (vi) In the conversion of intermediate 1 to intermediate 2 (figure 1) and in the conversion of intermediate 2 to intermediate 3 (figure 1), EEDQ is used as an amide coupling reagent. However, these reactions are not so limited by the specific reagent shown. One of skill in the art will recognize that amide coupling can be achieved with a number of different reagents, as reviewed in: Han SY and Kim YA The Recent Development of Peptide Coupling Reagents in Organic Synthesis. Tetrahedron 2004; 60: 2447-2467.

[0033] A therapeutic use of antibody conjugates of camptothecins, such as SN-38, which have nanomolar toxicities in vitro, compared to the sub-nanomolar to picomolar toxicities of ultratoxic chemotherapeutic agents such as calicheamicin, maytansinoids or MMAE, is described. The use of drugs that are not ultratoxic allows the use of antibody-drug linkers that do not require cellular internalization for free drug release, but rather allow some extracellular drug release. With the CL2A linker described herein, 50% of the conjugated drug is released within 24 hours, thus increasing the bioavailability of the drug by releasing it both extracellularly and intracellularly. Furthermore, the use of relatively non-toxic drugs allows the administration of higher doses of ADCs, leading to better therapeutic effects.

[0034] The antibody incorporated into the subject immunoconjugates may be of various isotypes, preferably human IgG1, IgG2, IgG3 or IgG4, most preferably comprising human IgG1 hinge and constant region sequences. The antibody or fragment thereof may be a chimeric, humanized, or fully human antibody or antigen-binding fragment thereof, such as semi-IgG4 antibodies, as described by van der Neut Kolfschoten et al. (Science 2007; 317: 1554-1557), or commercially available single domain antibodies (eg, nanobodies) (eg, ABLYNX®, Ghent, Belgium). More preferably, the antibody or fragment thereof may be designed or selected to comprise human constant region sequences belonging to specific allotypes, which may result in reduced immunogenicity when the immunoconjugate is administered to a human subject. Preferred allotypes for administration include a non-Glml (nG1m1) allotype, such as G1m3, G1m3.1, G1m3.2, or G1m3.1.2. More preferably, the allotype is selected from the group consisting of nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

[0035] The antibodies of use may bind to any disease-associated antigen known in the art. When the disease state is cancer, e.g. For example, many antigens expressed by or associated with with tumor cells, including but not limited to carbonic anhydrase IX, alpha-fetoprotein (AFP), a-actinin-4, A3, A33 antibody-specific antigen, 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-1a, colon-specific p-antigen (CSAp) , CEACAM5, CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1(TROP-2), EGP-2,ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt -3, folate receptor, G250 antigen, GAGE, gp100, GRO-p, HLA-DR, HM1,24, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, histone H2B, histone H3, histone H4, HMGB -1, hypoxy-inducible factor a (HIF-1), HSP70-2M, HST-2, la, IGF- 1R, IFN-y, IFN-a, IFN-p, IFN-A, 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, growth factor insulin-like-1(IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory 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, pancreatic cancer mucin, PD-1, PD-L1, 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-a, antigen Tn, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, fibronectin ED-B, WT-1, antigen 17-1A, complement factors C3, C3a, C3b, C5a, C5, a marker of angiogenesis, bc l-2,bcl-6, Kras, an oncogenic marker, and an oncogenic product (see, p. eg, 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 AFP, CEACAM5, CEACAM6, CSAp, EGP-1(TROP-2), AFP, MUC5ac, CD74, CD19, CD20, CD22, or HLA-DR.

[0036] Exemplary antibodies that can be used include, but are not limited to hR1 (anti-IGF-1 R, US Patent Application Serial Number 13/688,812, filed 11/29/12), hPAM4 (anti-MUC5ac, US Patent No. 7,282,567), hA20 (anti-CD20, US Patent No. 7,151,164), hA19 (anti-CD19, US Patent No. 7,109,304) , hIMMU31 (anti-AFP, US Patent No. 7,300,655), hLL1 (anti-CD74, US Patent No. 7,312,318), hLL2 (anti-CD22, US Patent No. 5,789,554 ), hRFB4 (anti-CD22, US Pat. No. 9,139,649), hMu-9 (anti-CSAp, US Pat. No. 7,387,773), hL243 (anti-HLA-DR, US Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, US Patent No. 6,676,924), hMN-15 (anti-CEACAM6, US Patent No. 8,287,865), hRS7 (anti-EGP-1 , US Patent No. 7,238,785), hMN-3 (anti-CEACAM6, US Patent No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, US Patent No. 7,138,496). 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). As used herein, the terms epratuzumab and hLL2 are interchangeable, as are the terms veltuzumab and hA20, hL243g4P, hL243gamma4P, and |Mm U-114. In specific preferred embodiments, the immunoconjugate can be a hMN-14-SN-38, hMN-3-SN-38, hMN-15-SN-38, hIMMU-31-SN-38, hRS7-SN-38, hR1- SN-38, hA20-SN-38, hPAM4-SN-38, hL243-SN-38, hLL1-SN-38, hRFB4-SN-38, hMu-9-SN-38, or hLL2-SN-38 conjugated to a CL2A linker.

[0037] Alternative antibodies of use include, but are not limited to abciximab (anti-glycoprotein Ilb/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 (anti-PD-1 receptor), nivolumab ( anti-PD-1 receptor), ipilimumab (anti-CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab (anti-CD125 ), obinutuzumab (GA101, anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, US Patent Application 11/983,372, filed as ATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab (anti-CD11a ), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-a 4 integrin), omalizumab (anti-IgE); anti-TNF-a 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), Benlysta (Human Genome Sciences); antibodies for Alzheimer's disease therapy such as Alz 50 (Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab, solanezumab, and infliximab; anti-fibrin antibodies such as 59D8, T2G1s, MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202 (Celgene), HuMax-CD38 (Genmab), or daratumumab (Johnson &Johnson); (anti-HIV antibodies such as P4/D10 (US Patent 8,333,971), Ab 75, Ab 76, Ab 77 (Paulik et al., 1999, Biochem Pharmacol 58: 1781-90), as well as anti- -HIV described and sold by Polymun (Vienna, Austria), also described in US Patent 5,831,034, US Patent 5,911,989, and Vcelar et al., AIDS 2007;21(16):2161- 2170 and Joos et al., Antimicrob Chemother Agents 2006;50(5):1773-9, and antibodies against pathogens such as CR6261 (anti-influenza), exbivirumab (anti-hepatitis B), felvizumab (anti-respiratory syncytial virus ), foravirumab (anti-rabies virus), motavizumab (anti-respiratory syncytial virus), palivizumab (anti-respiratory syncytial virus), panobacumab (anti-Pseudomonas), rafivirumab (anti-rabies virus), regavirumab (anti-cytomegalovirus ), sevirumab (anti-cytomegalovirus), tivirumab (anti-hepatitis B) and urtoxazumab (anti-E. coli).

[0038] Preferably, the antibody moiety is bound to at least one drug moiety, more preferably from 1 to about 5 drug moieties, alternatively from about 7 to 12 drug moieties. In various embodiments, the antibody moiety can be attached to 4 to 6 drug moieties, 6 to 8 drug moieties, or 7 to 8 drug moieties. of drug. The number of drug moieties per antibody moiety can be 1,2,3,4, 5, 6, 7, 8 or more.

[0039] The present description also relates to the use of the described methods and compositions for the treatment of a cancer, including, but not limited to non-Hodgkin's lymphomas, acute B-cell and chronic lymphoid leukemias, Burkitt's lymphoma, lymphoma Hodgkin's, 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, bone and skin cancers. Carcinomas may include carcinomas of the oral cavity, esophagus, gastrointestinal tract, pulmonary tract, lung, stomach, colon, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, brain, connective tissue, liver , gallbladder, urinary bladder, kidney, skin, central nervous system, and testicles.

[0040] Examples of diseases of autoimmune or immune dysfunction that can potentially be treated with the subject immunoconjugates include acute immune thrombocytopenia, chronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, pemphigus vulgaris, diabetes mellitus (eg, juvenile diabetes), Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis, sclerosis multiple, sarcoidosis, ulcerative colitis multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomatosis vulgaris, polymyositis vulgaris, polymyositis , mefigomondomyositis, polymyositis. Branous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, graft-versus-host disease (GVHD), organ transplant rejection, sepsis, sepsis, and inflammation.

[0041] In addition, the disclosed methods and compositions can be used to treat an infectious disease, for example diseases involving infection by pathogens such as bacteria, rickettsiae, mycoplasmas, protozoa, fungi, viruses, parasites, or other microbial agents. Examples include the human immunodeficiency virus (HIV) that causes AIDS, Mycobacterium tuberculosis, Streptococcus agalactiae, Methicillin- resistant Staphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningittopicmans, Treponema pallidum, Lyme disease spirochetes, West Nile virus, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, HCV-like virus human serum parvo, respiratory syncytial virus, varicella zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, murine leukemia, mumps virus, vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitis virus, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, reo virus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruz', Trypanosoma rhodesiensei, Trypanosoma brucecercai, Schistosoma mansoni, Schistosoma japanicum, Babesia tevisa bovis, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasmalaidlawii, M. salivarium, and M. pneumoniae. A review listing antibodies against infectious organisms (antitoxin and antiviral antibodies), as well as other targets, is found in Casadevall, Clin Immunol 1999; 93 (1): 5-15.

[0042] In certain examples involving the treatment of cancer, the drug conjugates may be used in combination with another therapeutic modality, such as surgery, radiation therapy, chemotherapy, naked antibody immunotherapy, radioimmunotherapy, immunomodulators, or vaccines. Similar combination therapies can be used in the treatment of other diseases susceptible to antibody moieties, such as autoimmune diseases. For example, camptothecin conjugates can be combined with TNF inhibitors, B-cell antibodies, interferons, interleukins, and other effective agents for the treatment of autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosis, Sjogren's syndrome, multiple sclerosis, vasculitis. , as well as type I diabetes (juvenile diabetes). These combination therapies may allow lower doses of each therapy to be administered in such combinations, thus reducing certain serious side effects and potentially reducing the courses of therapy required.

[0043] In infectious diseases, drug immunoconjugates can be combined with other therapeutic drugs, immunomodulators, naked MAbs, or vaccines (eg, MAbs against hepatitis, HIV, or papillomavirus, or immunogen-based vaccines). of these viruses, or kinase inhibitors, as in hepatitis B). Antibodies and antigen-based vaccines against these and other viral pathogens are known in the art and, in some cases, are already in commercial use. The development of anti-infective monoclonal antibodies has been recently reviewed by Reichert and Dewitz (Nat Rev Drug Discovery 2006; 5:191-195), who summarizes the priority pathogens against which naked antibody therapy has been applied, resulting in only 2 pathogens against which antibodies are in phase III clinical trials or are being marketed (respiratory syncytial virus and methicillin- resistant Staphylococcus aureus), with another 25 in clinical studies and 20 discontinued during clinical study. For combination therapy, the use of radioimmunotherapy for the treatment of infectious organisms is described, e.g. e.g., in the US Patent Nos. 4,925,648; 5,332,567; 5,439,665; 5,601,825; 5,609,846; 5,612,016; 6,120,768; 6,319,500; 6,458,933; 6,548,275; and in US Patent Application Publication Nos. 20020136690 and 20030103982.

[0044] The preferred optimal dosage of immunoconjugates may include a dose of between 3 mg/kg and 18 mg/kg, preferably administered weekly, twice weekly, every two weeks, or every three weeks. The optimal dosing schedule may include treatment cycles of two consecutive weeks of therapy followed by one, two, three, or four weeks off, or alternate weeks of therapy and rest, or one week of therapy followed by two, three, or four weeks. or three weeks of therapy followed by one, two, three, or four weeks off, or four weeks of therapy followed by one, two, three, or four weeks off, or five weeks of therapy followed by one, two, three, four, or five weeks off, or administration once every two weeks, once every three weeks, or once a month. The treatment may extend 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. Exemplary use levels 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. kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/ kg, 20 mg/kg, 22 mg/kg and 24 mg/kg. Preferred doses are 4, 6, 8, 9, 10, 12, 14, 16, or 18 mg/kg. The most preferred doses are 8 or 10 mg/kg. The person of ordinary skill will appreciate that a variety of factors, such as age, general health, specific organ function, or weight, as well as the effects of prior therapy on specific organ systems (eg, ., bone marrow) may be considered in selecting an optimal dose of immunoconjugate, and that the dose and/or frequency of administration may be increased or decreased during the course of therapy. The dose may be repeated as needed, with evidence of tumor shrinkage seen after as little as 4 to 8 doses. The optimized doses and administration schedules described herein show unexpected superior efficacy and reduced toxicity in human subjects, which could not have been predicted from studies in animal models. Surprisingly, the superior efficacy allows treatment of tumors previously found to be resistant to one or more standard anticancer therapies, including the parent compound, CPT-11, from which SN-38 is derived in vivo.

[0045] A surprising result with the compositions and methods described herein is the unexpected tolerability of high doses of antibody-drug conjugate, even with repeated infusions, with only relatively low-grade toxicities of nausea, vomiting, and diarrhea observed, as well as the manageable rash or neutropenia. Another surprising result is the lack of accumulation of the antibody-drug conjugate, unlike other products that have conjugated SN-38 with albumin, PEG, or other carriers. The lack of accumulation is associated with better tolerability and lack of serious toxicity even after repeated or increased doses. These surprising results allow optimization of dosage and administration schedule, with unexpectedly high efficacy and low toxicity. The claimed methods provide a tumor shrinkage, in individuals with previously resistant cancers, of 15% or more, preferably 20% or more, preferably 30% or more, more preferably 40% or more in size (measured by diameter). longer). One of skill in the art will appreciate that tumor size can be measured by a variety of different techniques, such as total tumor volume, maximum tumor size in any dimension, or a combination of size measurements in various dimensions. This can be with standard radiological procedures such as computed tomography, ultrasound and/or positron emission tomography. Means of measuring size are less important than observing a tendency for tumor size to decrease with immunoconjugate treatment, which preferably results in tumor removal.

[0046] While 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 extend the PK of the immunoconjugate in the blood, a continuous infusion, e.g. eg, through an indwelling catheter. Such devices are known in the art, such as HICKMAN®, BROVIAC®, or PORT-A-CATH® catheters (see, e.g., Skolnik et al., Ther DrugMonit 32:741-48, 2010) and any indwelling catheter. known can be used. A variety of continuous infusion pumps are also known in the art and any known infusion pump can be used. The dosage range for continuous infusion may be between 0.1 and 2.0 mg/kg per day. More preferably, these immunoconjugates can be administered by intravenous infusions over relatively short periods of 2 to 5 hours, more preferably 2 to 3 hours.

[0047] In particularly preferred examples, immunoconjugates and dosage schedules may be effective in patients resistant to standard therapies. For example, a mAb-CL2A-SN-38 immunoconjugate can be administered to a patient who has not responded to prior therapy with irinotecan, the parent agent of SN-38. Surprisingly, the irinotecan resistant patient may show a partial response to mAb-CL2A-SN-38. The ability of the immunoconjugate to specifically target tumor tissue can overcome tumor resistance by improving targeting and improved delivery of the therapeutic agent. Combinations of different SN-38 immunoconjugates, or SN-38-antibody conjugates in combination with an antibody conjugated to a radionuclide, toxin, or other drug, may provide even further improved efficacy and/or reduced toxicity. A specific preferred subject may be a metastatic colon cancer patient, a triple negative breast cancer patient, a HER+, ER+, progesterone+ breast cancer patient, a metastatic non-small cell lung cancer (NSCLC) patient, a patient with metastatic pancreatic cancer, a patient with metastatic renal cell carcinoma, a patient with metastatic gastric cancer, a patient with metastatic prostate cancer, or a patient with metastatic small cell lung cancer. In different embodiments, the therapy it may be suitable for treating first-line, second-line, third-line or other patients.

[0048] The invention as defined in the claim relates to an improved method for preparing CL2A-SN38 conjugates and CL2A-SN38 antibody immunoconjugates on a large scale, with better yield and/or efficiency. The synthetic scheme is shown in FIG. 1. Preferably, the maleimide group at the end of the CL2a linker reacts with sulfhydryl side chains on reduced cysteine residues in an antibody moiety or other target peptide or protein, although other methods of attaching the CL2A moiety to the antibody moiety and they can be worn. In a further preferred embodiment, immunoconjugates prepared from the CL2A-SN-38 moiety are purified by tangential flow filtration, thus avoiding cumbersome chromatography on size exclusion and hydrophobic interaction columns and allowing high protein recovery after filtration. the purification.

BRIEF DESCRIPTION OF THE FIGURES

[0049]

FIG. 1. New scheme to improve large-scale production of CL2A-SN-38.

FIG. 2. In vivo therapy of athymic nude mice, carriers of Capan 1 human pancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 3. In vivo therapy of athymic nude mice, bearing human pancreatic carcinoma BxPC3, with MAb-CL2A-SN-38 conjugates.

FIG. 4. In vivo therapy of athymic nude mice, bearing LS174T human colon carcinoma, with hMN-14-CL2A-SN-38 conjugate.

FIG. 5A. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing human non-small cell lung tumor xenografts. Mice bearing Calu-3 tumors (n=5-7) were injected with hRS7-CL2-SN-38 every 4 days for a total of 4 injections (q4dx4). All ADCs and controls were administered in the amounts indicated (expressed as amount of SN-38 per dose; long arrows = conjugate injections, short arrows = irinotecan injections).

FIG. 5B. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing human colorectal tumor xenografts. COLO 205 tumor-bearing mice (n=5) were injected 8 times (q4dx8) with the ADC or every 2 days for a total of 5 injections (q2dx5) with the irinotecan MTD. All ADCs and controls were administered in the amounts indicated (expressed as amount of SN-38 per dose; long arrows = conjugate injections, short arrows = irinotecan injections).

FIG. 5C. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing human pancreatic cancer xenografts. Capan-1 tumor-bearing mice (n=10) (n=10) were treated twice weekly for 4 weeks with the indicated agents. All ADCs and controls were administered in the amounts indicated (expressed as amount of SN-38 per dose; long arrows = conjugate injections, short arrows = irinotecan injections).

FIG. 5 D. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing human pancreatic cancer xenografts. BxPC-3 tumor-bearing mice (n=10) were treated twice weekly for 4 weeks with the indicated agents. All ADCs and controls were administered in the amounts indicated (expressed as amount of SN-38 per dose; long arrows = conjugate injections, short arrows = irinotecan injections).

FIG. 5E. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing human squamous cell lung carcinoma xenografts. In addition to ADC administered twice weekly for 4 weeks, SK-MES-1 tumor-bearing mice (n=8) received the MTD of CPT-11 (q2dx5). All ADCs and controls were administered in the amounts indicated (expressed as amount of SN-38 per dose; long arrows = conjugate injections, short arrows = irinotecan injections).

FIG. 6. Comparative efficacy of epratuzumab (Emab)-SN-38 and veltuzumab (Vmab)-SN-38 conjugates in the subcutaneous Ramos model. Nude mice (n=10 per group) with tumors averaging approximately 0.35 cm3 (0.20-0.55 cm3) were administered 0.25 or 0.5 mg of each conjugate twice weekly for 4 days. weeks.

FIG. 7A. Specificity of anti-CD22-SN-38 Emab conjugate (solid line) versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) in nude mice bearing subcutaneous Ramos tumors. Animals were dosed twice weekly with 75 pg of each conjugate per dose (54.5 pg/kg SN-38, based on a mean weight of 22 g) intraperitoneally for 4 weeks. Survival based on time to progression (TTP) up to 3.0 cm3, with tumors starting at a mean size of 0.4 cm3. P-values comparing median survival (shown) for Emab-SN-38 conjugate with Lmab-SN-38 are shown in each panel.

FIG. 7B. Specificity of anti-CD22-SN-38 Emab conjugate (solid line) versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) in nude mice bearing subcutaneous Ramos tumors. Animals were dosed twice weekly with 125 pg of each conjugate per dose (91 pg/kg SN-38, based on a mean weight of 22 g) intraperitoneally for 4 weeks. Survival based on time to progression (TTP) up to 3.0 cm3, with tumors starting at a mean size of 0.4 cm3. P-values comparing median survival (shown) for Emab-SN-38 conjugate with Lmab-SN-38 are shown in each panel.

FIG. 7C. Specificity of the anti-CD22-SN-38 Emab conjugate (solid line) against an irrelevant conjugate of labetuzumab (Lmab)-SN-38 (dashed line) in nude mice bearing subcutaneous Ramos tumors. Animals were dosed twice weekly with 250 pg of each conjugate per dose (182 pg/kg SN-38, based on a mean weight of 22 g) intraperitoneally for 4 weeks. Survival based on time to progression (TTP) up to 3.0 cm3, with tumors starting at a mean size of 0.4 cm3. P-values comparing median survival (shown) for Emab-SN-38 conjugate with Lmab-SN-38 are shown in each panel. Survival curves (solid gray) are also shown for another group of animals that received weekly intraperitoneal injections of irinotecan (6.5 pg/dose; SN-38 equivalent to approximately the same as the 250 pg dose of the EMAB-SN-38). 38 conjugate).

FIG. 8. Patient's prior treatment history, prior to administering IMMU-130 (labetuzumab-NS-38). Previous treatment included colectomy/hepatectomy (partial lobe) for stage IV RCC, radiofrequency ablation therapy of liver metastases, wedge resection of lung metastases, and chemotherapy with irinotecan/oxaliplatin, Folfirinox, Folfirinox bevacizumab, bevacizumab 5-FU /leucovorin, FolFiri, Folfiri cetuximab and cetuximab alone. The patient received 16 mg/kg doses of IMMU-132 by slow IV infusion every two weeks for a total of 17 treatment doses.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0050] In the description that follows, a number of terms are used and the following definitions are provided to facilitate understanding of the claimed subject matter. Terms not expressly defined herein are used in accordance with their simple and ordinary meanings.

[0051] Unless otherwise specified, one or ones means "one or more".

[0052] The term approximately is 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.

[0053] An antibody, as used herein, refers to a full-length (ie, naturally occurring or formed by normal immunoglobulin from recombinatorial processes of gene fragments) immunoglobulin molecule (eg, an IgG antibody) or an antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody or antibody fragment may be conjugated or otherwise derivatized within the scope of the claimed subject matter. Such antibodies include, but are not limited to IgG1, IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes.

[0054] An antibody fragment is a portion of an antibody 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 half molecules cited above (van der Neut Kolfschoten et al. (Science 2007; 317 (Sep 14): 1554-1557). A commercially available form of single-domain antibody, termed a nanobody (ABLYNX ®, Ghent, Belgium), is discussed in more detail below. Regardless of structure, an antibody fragment in use binds the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes proteins synthetic or genetically engineered antibodies that act like an antibody by binding to a specific antigen to form a complex For example, antibody fragments include isolated fragments consisting of the variable regions, such as "Fv" fragments consisting of the variable regions Variable regions of heavy and light chains, recombinant single-chain polypeptide molecules in which the heavy and light variable regions are connected by a peptide linker ("scFv proteins") and minimal recognition units consisting of the amino acid residues that mimic the region. hypervariable, such as CDRs. Fv fragments can be constructed in different ways to produce multivalent and/or multispecific binding forms. In the case of multivalents, they have more than one binding site against the specific epitope, while with the multispecific forms, more than one epitope (either from the same antigen or against an antigen and a different antigen) binds.

[0055] A naked antibody is generally a whole antibody that is not conjugated to a therapeutic agent. This is so because the Fc portion of the antibody molecule provides effector or immunological functions, such as complement fixation and ADCC (antibody-dependent cellular cytotoxicity), which trigger mechanisms that can result in cell lysis. However, the Fc portion may not be necessary for the therapeutic function of the antibody, but for other mechanisms, such as apoptosis, anti-angiogenesis, anti-metastatic activity, anti-adhesion activity, such as inhibition of heterotypic or homotypic adhesion and interference. in signaling pathways, they can come into play and interfere with disease progression. Naked antibodies include polyclonal and monoclonal antibodies, and fragments thereof, including murine antibodies, as well as certain recombinant antibodies, such as chimeric, humanized, or human antibodies, and fragments thereof. As used herein, "naked" is synonymous with "unconjugated" and means not bound or conjugated to a therapeutic agent.

[0056] A chimeric antibody is a recombinant protein that contains the variable domains of both the heavy chain and light chains of antibody, including the complementarity determining regions (CDRs) of a antibody derived from a species, preferably a rodent antibody, more preferably a murine antibody, while the constant domains of the antibody molecule are derived from those of a human antibody. For veterinary applications, the chimeric antibody constant domains can be derived from those of other species, such as primates, cats or dogs.

[0057] A humanized antibody is a recombinant protein wherein the CDRs of an antibody of one species; for example, a murine antibody, are transferred from the murine antibody heavy and light variable chains to human heavy and light variable domains (framework regions). The constant domains of the antibody molecule are derived from those of a human antibody. In some cases, specific residues in the framework region of the humanized antibody, particularly those in contact with or near the CDR sequences, may be modified, e.g. eg, be replaced with corresponding residues from the original murine, rodent, subhuman primate, or other antibody.

[0058] A human antibody is an antibody obtained, e.g. eg, from transgenic mice that have been "modified" to produce human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for various antigens, and the mice can be used to produce hybridomas that secrete 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 methods, as well as phage display technology, all of which are known in the art. See, p. eg, McCafferty et al., Nature 348: 552-553 (1990) for the production of human antibodies and fragments thereof in vitro, from immunoglobulin variable domain gene repertoires of unimmunized donors. In this technique, variable domain antibody genes are cloned in frame into a major or minor coat protein gene of a filamentous bacteriophage and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B cell. Phage display can be done in a variety of formats, for review see p. eg, Johnson and Chiswell, Current Opinion in Structural Biology 3: 5564-571 (1993). Human antibodies can also be generated by activated B cells in vitro. See US Patent Nos. 5,567,610 and 5,229,275.

[0059] Infectious Diseases as used herein are diseases involving infection by pathogens such as bacteria, rickettsiae, mycoplasmas, protozoa, fungi, viruses, parasites, or other microbial agents. Examples include the human immunodeficiency virus (HIV) that causes AIDS, Mycobacterium tuberculosis, Streptococcus agalactiae, Methicillin-resistant Staphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhosae, Neisseria meningittocotipus, Treponema pallidum, Lyme disease spirochetes, West Nile virus, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, HCV-like virus human serum parvo, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, virus murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitis virus, wart virus , bluetongue virus, Sendai virus, feline leukemia virus, reo virus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucecercai, Schistosoma mansoni, Schistosoma japanicum, Onmerchoia bovis, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma corti , M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, and M. pneumoniae. A review listing antibodies against infectious organisms (antitoxin and antiviral antibodies), as well as other targets, is found in Casadevall, Clin Immunol 1999; 93 (1): 5-15.

[0060] A therapeutic agent is a molecule or atom that is administered separately, simultaneously or sequentially with a binding moiety, e.g. eg, an antibody or antibody fragment, and is useful in the treatment of a disease. Examples of therapeutic agents include, but are not limited to, antibodies, antibody fragments, conjugates, drugs, cytotoxic agents, proapoptotic agents, toxins, nucleases (including DNases and RNases), hormones, immunomodulators, chelators, boron compounds, photoactive agents, or dyes , radioisotopes or radionuclides, oligonucleotides, RNA interference, peptides, anti-angiogenic agents, chemotherapeutic agents, cycokines, chemokines, prodrugs, enzymes, binding proteins or peptides, or combinations thereof.

[0061] An immunoconjugate is an antibody, antibody fragment, or other antibody moiety conjugated to a therapeutic agent. As used herein, the terms "conjugate" and "immunoconjugate" are used interchangeably.

[0062] As used herein, the term "antibody fusion protein" is a recombinantly produced antigen-binding molecule in which one or more natural antibodies, single-chain antibodies, or antibody fragments are linked to another moiety. , such as a protein or peptide, a toxin, a cytokine, a hormone etc In certain preferred embodiments, the fusion protein may comprise two or more of the same or different antibodies, antibody fragments, or single-chain antibodies fused together, which may bind to the same epitope, different epitopes on the same antigen, or different antigens.

[0063] An immunomodulator is a therapeutic agent that, when present, alters, suppresses, or stimulates the body's immune system. Typically, an immunomodulator in use stimulates immune cells to proliferate or activate in an immune response cascade, such as macrophages, dendritic cells, B cells, and/or T cells. However, in some cases, an immunomodulator may suppress proliferation or activation of immune cells, as in the therapeutic treatment of autoimmune diseases. An example of an immunomodulator as described herein is a cytokine, which is a small soluble protein of approximately 5-20 kDa that is released by a cell population (eg, primed T cells) on contact with specific antigens, and that acts as an intercellular mediator between cells. As will be understood by those skilled in the art, examples of cytokines include lymphokines, monokines, interleukins, and various related signaling molecules, such as tumor necrosis factor (TNF) and interferons. Chemokines are a subset of cytokines. Certain interleukins and interferons are examples of cytokines that stimulate the proliferation of T cells or other immune cells.

[0064] CPT is an abbreviation for camptothecin. As used in the present application, CPT represents camptothecin itself or an analog or derivative of camptothecin. The structures of camptothecin and some of its analogs, with numbering indicated and rings labeled AE, are given in formula 1 in Diagram 1 below.

Diagram 1

[0065]

CPT: R1 = R2 = R3 = H

10-hydroxy-CPT: R1 = OH; R2 = R3 = H CPT-11: R1 =

R2 = ethyl; R3 = H

SN-38: R1 = OH; R2 = ethyl; R3 = H

Topotecan: R1 = OH; R2=H; R3 = CH2-N(CH3)2

Abbreviations:

[0066]

DCA - dichloroacetic acid

Fmoc - fluorenylmethyloxycarbonyl chloride

EEDQ - 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

MCC-yne - 4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide

MMT - monomethoxytrityl

PABOH - p-aminobenzyl alcohol

PEG - polyethylene glycol

TBAF - tetra-n-butylammonium fluoride

TBDMS - tert-butyldimethylsilyl

Camptothecin conjugates

[0067] Non-limiting methods and compositions for preparing immunoconjugates comprising a camptothecin therapeutic agent linked to an antigen-binding antibody or antibody fragment are described below. In preferred embodiments of the invention as defined in the claims, the solubility of the drug is enhanced by placing a defined polyethylene glycol (PEG) moiety (i.e., a PEG containing a defined number of monomeric units) between the drug and the antibody, wherein the defined PEG is a low molecular weight PEG, preferably containing 1 -30 monomeric units, more preferably containing 1-12 monomeric units, most preferably containing 8 monomeric units.

[0068] Preferably, a first linker connects the drug at one end and may end with an acetylene or azide group at the other end. This first linker may comprise a defined PEG moiety with an azide or acetylene group at one end and a different reactive group, such as a carboxylic acid or hydroxyl group, at the other end. Said bifunctional defined PEG can bind to the amine group of an amino alcohol, and the hydroxyl group of the latter can bind to the hydroxyl group of the drug in carbonate form. Alternatively, the non-azide (or acetylene) moiety of said defined bifunctional PEG may be attached to the N-terminus of an L-amino acid or a polypeptide, with the C-terminus attached to the amino group of the amino alcohol, and the hydroxy group of the latter it can be attached to the hydroxyl group of the drug in the form of carbonate or carbamate, respectively.

[0069] A second linker, comprising a coupling antibody group and a complementary reactive group on the azide (or acetylene group) of the first linker, namely acetylene (or azide), can react with the drug (first linker) conjugate via acetylene-azide cycloaddition reaction to provide a final bifunctional drug product that is useful for conjugating with disease-directed antibodies. The coupling group of the antibody is preferably a thiol or a thiol-reactive group.

[0070] Methods for the selective regeneration of the 10-hydroxyl group in the presence of the C-20 carbonate in drug linker precursor preparations 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 t-butyldimethylsilyl or t-butyldiphenylsilyl, and these can be deprotected by tetrabutylammonium fluoride prior to attachment of the derivatized drug to an antibody coupling moiety. The 10-hydroxyl group of CPT analogs is alternatively protected as an ester or carbonate, other than "BOC", so that the bifunctional CPT is conjugated to an antibody without prior deprotection of this protecting group. The protecting group can be easily deprotected under physiological pH conditions after administration of the bioconjugate.

[0071] In the acetylene-azide coupling, which is referred to as "click chemistry", the azide moiety may be in L2 with the acetylene moiety in L3. Alternatively, L2 may contain acetylene, while L3 contains azide. "Click chemistry" refers to a copper (+1) catalyzed cycloaddition reaction between an acetylene moiety and an azide moiety (Kolb HC and Sharpless KB, Drug Discov Today 2003; 8: 1128-37), although alternative forms exist. of click chemistry. known and can be used. The reaction can use a mixture of cuprous bromide and triphenylphosphine to allow highly efficient coupling in non-polar organic solvents such as dichloromethane. However, as described below, triphosphine is disadvantageous for certain purposes and alternative methods can be used to facilitate the click coupling step. The advantage of click chemistry is that it is chemoselective and complements other well-known conjugation chemistries, such as the thiol-maleimide reaction. In the following discussion, when a conjugate comprises an antibody or antibody fragment, it may be substituted with another type of binding moiety, such as a targeting peptide.

[0072] An example of the present description is directed to a conjugate of a drug derivative and an antibody of the general formula 2,

MAB-[L2]-[L1 ]-[AA]m-[A']-drug (2)

where MAb is a disease directed antibody; L2 is a component of the crosslinker comprising an antibody coupling moiety and one or more acetylene (or azide) groups; L1 comprises a PEG defined with azide (or acetylene) at one end, complementary to the acetylene (or azide) moiety in L2, and a reactive group such as carboxylic acid or hydroxyl group at the other end; AA is an L-amino acid; m is an integer with values of 0, 1.2, 3 or 4 (preferably 1); and A' is an additional spacer, selected from the group of substituted or unsubstituted ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, or ethylenediamine. The L amino acids of 'AA' 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 valine. If the A' group contains hydroxyl, it is bound to the hydroxyl group or amino group of the drug in the form of carbonate or carbamate, respectively.

[0073] In a preferred example of formula 2, 'A' of general formula 2 is A-OH, where A-OH is a folding moiety such as 4-aminobenzyl alcohol or a substituted 4-aminobenzyl alcohol, substituted with a C1-C10 alkyl group at the benzylic position, and the second, through its amino group, is attached to an L-amino acid or a polypeptide comprising up to four amino acid L acid residues; wherein the n-terminus is attached to a cross-linking agent which terminates in the linking group of the antibody moiety.

[0074] A preferred example is the following, wherein the A-OH embodiment of A' of general formula (2) is derived from substituted 4-aminobenzyl alcohol, and 'AA' is composed of an L-amino acid unique with m = 1 in the general formula (2), and the drug is exemplified by SN-38. The structure is depicted below (formula 3, designated MAb-CLX-SN-38). A single AA amino acid is 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 substituent R on the 4-aminobenzyl alcohol moiety (A-OH embodiment of A') is hydrogen or an alkyl group selected from C1-C10 alkyl groups.

[0075] According to the invention as defined in the claims, MAb-CLX-SN-38 of formula 3 is such that the single AA amino acid is L-lysine and R=H, and the drug is exemplified by SN- 38 (formula 4; designated MAb-CL2A-SN-38).

[0076] In another example, the L1 component of the conjugate contains a defined polyethylene glycol (PEG) spacer with 1-30 repeating monomer units. In a further preferred embodiment, PEG is defined PEG with 1-12 (more preferably 8) repeating monomer units. The introduction of PEG may involve the use of heterobifunctionalized PEG derivatives that are commercially available. The heterobifunctional PEG may contain an azide or an acetylene group. An example of a heterobifunctional defines PEG containing 8 monomeric repeating units, 'NHS' being succinimidyl, is given below in formula 5:

[0077] The production process of CL2A-SN-38 according to the invention as defended in the claimsis as shown in FIG. 1. Certain significant process changes have been made in the preparation of CL2A-SN-38 used to produce the MAb-CL2A-SN-38 immunoconjugate. These changes in the procedures described, e.g. eg, in US Pat. No. 7,999,083, which are not obvious to one skilled in the art, comprise the following changes.

1) Although hexane was previously used to precipitate Lys(MMT)-PABOH (intermediate 2 in FIG. 1), hexane was substituted for heptane as a safer alternative. Residual unremoved diethylamine was assayed by NMR spectroscopy. If the presence of diethylamine was detected, the material was further purified to remove diethylamine, avoiding the reduction in yield caused by the presence of diethylamine in intermediate 2.

2) In the preparation of 10-0-TBDMS-SN-38 (intermediate 4 in FIG. 1) from SN-38, dimethylformamide (DMF) which was previously used as a solvent for the reaction was replaced by dichloromethane (DCM) , thus facilitating the removal of the solvent after the reaction. The change from DMF to DCM was done to facilitate scale-up as well as to reduce the product yield that was occasionally observed with the reaction in DMF. Protection of the hydroxyl group with tert-butyldimethylsilyl chloride is usually done using DMF as a solvent, although other solvents have been used (Greene TW and Wuts PGM, Protecting Groups in Organic Synthesis, John Wiley & Sons, Inc., 1999; pp 127 - 132). In the context of SN-38, the use of DMF was preferred as the material is poorly soluble in DCM. The high yield of product formation in DCM medium was unexpected and could not have been anticipated.

3) In the conversion of 10-0-TBDMS-SN-38 to its chloroformate-reactive intermediate 5 in FIG. 1, triphosgene was added to the dichloromethane solution in portions rather than as a batch. This preserved performance, while avoiding a potentially dangerous rise in reaction temperatures in large-scale manufacturing.

4) In the cycloaddition reaction between azido-PEG-Lys (MMT)-PABO-CO-20-0-SN-38 (intermediate 7 in FIG. 1) and MCC-yne (intermediate 8 in FIG. 1) to result in the penultimate intermediate, MCC-PEG-Lys (MMT)PABOCO- 20-0-SN-38 (intermediate 9 in FIG. 1), the product was found to be unstable if the reaction mixture was first treated with EDTA extraction to remove the copper salt, which leads to reduced performance. Surprisingly, it was also found that if chromatography of the reaction mixture was performed first, followed by EDTA extraction to remove residual copper salt that was not completely trapped in the silica gel column, the product was stable. This process change also led to yield improvements by avoiding the formation of unwanted by-products. It was not expected to improve the stability of intermediate 9 prior to purification by simply reversing the sequence of operations, ie, performing chromatography first followed by EDTA extraction.

5) In the same reaction as described in point 4 above, an improved yield was obtained when the reaction time was reduced to 14h, instead of 18-24h.

[0078] Additional improvements to the synthetic protocol are incorporated in the Figure. 1, which were not described in Priority US Patent 9,107,960. These changes to the protocol, which are not obvious to one skilled in the art, comprise the following changes.

6) The two-step conversion of starting material 1 to intermediate 2 originally involved isolation of the intermediate from the first step. This process was telescopic in such a way that isolation of the intermediate between starting material 1 and intermediate 2 was no longer necessary. Other process improvements for this two-step conversion involved a large reduction in the amount of volatile diethylamine used, improved safety, and easier subsequent removal during the isolation of Intermediate 2.

7) The two-step preparation of MCC-yne (intermediate 8), not shown in the schematic, originally involved chromatographic purification after each step; The reaction conditions for both stages were changed, allowing the isolation of both intermediate 8 and its precursor by crystallization or precipitation, without resorting to chromatography.

8) The two-step conversion of Intermediate 3 to Intermediate 7 originally involved the isolation of Intermediate 6. This process was simplified so that the isolation of Intermediate 6 was no longer necessary. Other process improvements for this two-step conversion included reduction in the number of phase splits after each step (resulting in higher yields and reduction in resulting emulsions) as well as reduction in the amount of TBAF required. for deprotection, resulting in an improved purity profile. Chromatographic conditions for the isolation of intermediate 7 were also improved to ensure adequate separation of impurities and complete recovery of product from the column.

9) The original conditions in the conversion of intermediate 7 to intermediate 9 involved the use of triphenylphosphine (PPh3) together with copper (I) bromide; these conditions contributed to the slow degradation of intermediate 9 and were therefore replaced by conditions [copper(II) sulfate, sodium ascorbate, and 2,6-lutidine] that provided much better solution stability of intermediate 9 and much better overall quality of the CL2A-SN-38 end product.

[0079] Changes to the synthesis protocol described above resulted in substantial improvements in yield and production efficiency, as well as decreased presence of byproduct contamination in the final product and in various intermediate steps. The decrease in contaminants also allowed the simplification or elimination of certain purification steps in intermediate synthetic reactions, also improving yield and efficiency.

[0080] After the CL2A-SN-38 product is formed, it can be conjugated to a target molecule, such as an antibody that binds to another tumor-associated or disease-associated antigen. In certain embodiments, when the bifunctional drug contains a thiol-reactive moiety as an antibody-binding group, the thiols of the antibody to be labeled may be generated from the side chains of the lysine groups of the antibody, rather than reduced cysteine residues, by the use of a thiolating reagent. Methods for introducing thiol groups into antibodies by modification of lysine mAb groups are well known (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 the antibody using the reducing agent, tris-(2-carboxyethyl)phosphine (TCEP), can generate 7 to 10 thiols in the antibody; which has the advantage of incorporating multiple drug moieties into the interchain region of the MAb away from the antigen-binding region. Furthermore, after reduction with TCEP, no prior purification of the reduced antibody is necessary, and the disulfide-reduced antibody is conjugated to the thiol reagent CL2A-SN-38 in situ.

[0081] In another preferred embodiment, the antibody and CL2A-SN-38 conjugates are purified by the tangential flow filtration (TFF) method using a 50,000 Da molecular weight membrane cut-off using 25 to 30 diafiltration volumes of the buffer of conjugate formulation to purify hundreds of grams of the conjugates. This method avoids the need to employ costly and cumbersome chromatographic purifications on hydrophobic and size exclusion chromatography columns.

[0082] In yet another embodiment, the conjugates are formulated in Good's biological buffers at a pH of 6 to 7.0, and lyophilized for storage. Preferably, Good's buffer is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)piperazine-1 -ethanesulfonic acid (HEPES) and 1,4-piperazinediethanesulfonic acid (PIPES), in the pH range of 6-7, preferably in the pH range of 6.5 to 7, and in a buffer concentration of 10-100 mM, preferably 25 mM. The most preferred formulation buffer is 25 mM MES, pH 6.5.

[0083] In a further embodiment, the purified conjugates are combined with excipients such as trehalose and polysorbate 80, lyophilized, and stored as lyophilisates in the temperature range of -20°C to 8°C.

General antibody techniques

[0084] Techniques for preparing monoclonal antibodies against virtually any target antigen are well known in the art. See, p. eg, Kohler 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 obtained by injecting mice with a composition comprising an antigen, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies against antigen, culturing the clones that produce antibodies against the antigen, and isolating the antibodies from the hybridoma cultures. Where antibodies are desired for use in human therapy or diagnosis, it will be understood by one skilled in the art that the corresponding human antigenic protein or peptide is preferably used to induce antibody production.

[0085] Various techniques, such as the production of chimeric or humanized antibodies, may involve antibody cloning procedures and construction. The Vk (Variable Light Chain) and ^Vh (Variable Heavy Chain) antigen-binding sequences for an antibody of interest can be obtained by a variety of molecular cloning procedures, such as RT-PCR, 5'-RACe, and RNA library screening. cDNA. Antibody V genes from a cell expressing a murine antibody can be cloned by PCR amplification and sequenced. To confirm their authenticity, the ^{cloned V l} and V ^h genes can be expressed in cell culture as a chimeric Ab as described by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86:3833 (1989) ). Based on the V gene sequences, a humanized antibody can be designed and constructed as described by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

[0086] The cDNA can be prepared from any known hybridoma line or transfected cell line that produces a murine antibody by general molecular cloning techniques (Sambrook et al., Molecular Cloning, Laboratory Manual, 2nd Edition (1989)) . The Vk sequence for the antibody can be amplified using the VK1BACK and VK1FOR primers (Orlandi et al., 1989) or the extended primer set described by Leung et al. (BioTechniques, 15: 286 (1993)). V sequences can be amplified using ^h primer pair VH1BACK / VH1FOR (Orlandi et to the., 1989) or the primers which hybridize to the constant region of murine IgG described by Leung et to the. (Hybridoma, 13:469 (1994)). The V ^h umanizados genes may be constructed by a combination of synthesis of long oligonucleotides and template PCR amplification as described by Leung et to the. (Mol. Immunol., 32: 1413 (1995)).

[0087] The PCR products for Vk can be subcloned into a staging vector, such as a pBR327-based staging vector, VKPBR, containing an Ig promoter, a signal peptide sequence, and convenient restriction sites. PCR products for V ^H can be subcloned into a staging vector similar as the pBluescript - based VHpBS I. The expression cassettes containing the Vk sequences and V ^h together with the promoter and sequences of the signal peptide may be cleaved from VKPBR and VHpBS and ligated into appropriate expression vectors, such as pKh and pG1g, respectively (Leung et to the., Hybridoma , 13: 469 (1994)). Expression vectors can be co-transfected into an appropriate cell and supernatant fluids monitored for production of a chimeric, humanized, or human antibody. Alternatively, expression cassettes Vk and V ^h can be cleaved and subcloned into a single expression vector, such as pdHL2, as described by Gillies et to the. (J. Immunol. Methods 125:191 (1989) and has also been shown in Losman et al., Cancer, 80: 2660 (1997)).

[0088] In an alternative embodiment, expression vectors can be transfected into host cells that have been pre-adapted for transfection, growth, and expression in serum-free medium. Exemplary cell lines that can be used include the Sp/EEE, Sp/ESF, and Sp/ESF-X cell lines (see, eg, US Patent Nos. 7,531,327; 7,537,930 and 7,608,425). These exemplary cell lines are based on the Sp2/0 myeloma cell line, transfected with a mutant Bcl-EEE gene, exposed to methotrexate to amplify the transfected gene sequences, and preadapted to the cell line without serum for protein expression.

Chimeric and humanized antibodies

[0089] A chimeric antibody is a recombinant protein in which the variable regions of a human antibody have been replaced by the variable regions of, e.g. eg, a mouse antibody, including the complementarity determining regions (CDRs) of the mouse antibody. Chimeric antibodies exhibit decreased 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).

[0090] A monoclonal chimeric antibody can be humanized by transferring the mouse CDRs of the mouse immunoglobulin heavy and light variable chains into the corresponding variable domains of a human antibody. The mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced with human FR sequences. To preserve the stability and antigenic specificity of the humanized monoclonal, one or more human FR residues may be replaced by homologous mouse residues. Humanized monoclonal antibodies can be used for therapeutic treatment of subjects. Techniques for the production of humanized monoclonal antibodies are well known in the art. (See, e.g., Jones et al., 1986, Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen et al., 1988, Science, 239:1534; Carter et al. ., 1992, Proc. Nat'l Acad. Sci. USA, 89: 4285; Sandhu, Crit. Rev. Biotech., 1992, 12: 437; Tempest et al., 1991, Biotechnology 9: 266; Singer et al., J. Immun, 1993, 150: 2844).

[0091] Other embodiments may refer to non-human primate antibodies. General techniques for generating therapeutically useful antibodies in baboons can be found, e.g. in Goldenberg et al., WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46:310 (1990). In another embodiment, an antibody can be a human monoclonal antibody. Such antibodies can be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigen challenge, as described below.

human antibodies

[0092] Methods for producing fully human antibodies using either combinatorial approaches or transgenic animals transformed with human immunoglobulin loci are known in the art (eg, 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). Such fully human antibodies are expected to show even fewer side effects than chimeric or humanized antibodies and to function in vivo as essentially endogenous human antibodies. In certain embodiments, the claimed methods and procedures may use human antibodies produced by such techniques.

[0093] In an alternative, the phage display technique can be used to generate human antibodies ( eg, DantasBarbosa et al, 2005, Genet Mol Res 4:126-40). Human antibodies can be generated from normal humans or from humans with a particular disease state, such as cancer (Dantas-Barbosa et al., 2005). The advantage of constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.

[0094] In a non-limiting example of this methodology, Dantas-Barbosa et al. (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Total RNA was generally obtained from circulating blood lymphocytes ( Id.). Recombinant Fabs were cloned from the m, y, and k chain antibody repertoires and inserted into a phage display library ( Id.). The RNAs were converted to cDNA and used to make Fab cDNA libraries using primers specific against heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222: 581-97). Library construction was performed according to Andris-Widhopf et al. (2000, in: Phage Display Laboratory Manual, Barbas et al. (Eds), 1st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp 9.1-9.22). The final Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to make the phage display library. Such libraries can be screened by standard phage display methods. One of skill in the art will appreciate that this technique is by way of example only and any known method for making and screening human antibodies or antibody fragments by phage display can be used.

[0095] In another alternative, transgenic animals that have been genetically engineered to produce human antibodies can be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols as discussed above. Green et al., Nature Genet describe methods for obtaining human antibodies from transgenic mice. 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 the XenoMouse® (eg, Green et al., 1999, J. Immunol. Methods 231:11-23) from Abgenix (Fremont, CA). In this and similar animals, the mouse antibody genes have been inactivated and replaced with functional human antibody genes, while the rest of the mouse immune system remains intact.

[0096] The XenoMouse® was transformed with germline-configured YACs (yeast artificial chromosomes) containing portions of the human IgH and Ig kappa loci, including most of the variable region sequences, along with accessory genes and regulatory sequences . The human variable region repertoire can be used to generate antibody-producing B cells, which can be processed into hybridomas by known techniques. Immunization with a target antigen will produce human antibodies by the normal immune response, which can be harvested and/or produced by standard techniques discussed above. A variety of strains of such transgenic mice are available, each of which is capable of producing a different class of antibody. 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 appreciate that the claimed compositions and methods are not limited to the use of the specific system, but can use any transgenic animal that has been genetically modified to produce human antibodies.

Production of antibody fragments

[0097] Antibody fragments can be obtained, e.g. by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from disulfide bond cleavage, to produce monovalent 3.5S Fab' fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and one Fc fragment. Exemplary methods for producing antibody fragments are described in US Patent No. 4,036,945; US Patent No. 4,331,647; 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).

[0098] Other antibody cleavage methods, such as separation of heavy chains to form monovalent heavy-light chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, provided that the fragments bind to the antigen that is recognized by intact antibody. For example, Fv fragments comprise an association of V ^h and V ^l chains. This association may be non-covalent, as described in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659. Alternatively, the variable chains may be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See Sandhu, 1992, Crit. Rev. Biotech., 12: 437.

[0099] Preferably, Fv fragments comprise V ^h chains and V ^L connected by a peptide linker. These single-chain antigen-binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the 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 peptide linker linking the two V domains. Methods for producing 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; US Patent No. 4,946,778; Pack et al., 1993, Bio/Technology, 11:1271 and Sandhu, 1992, Crit. Rev. Biotech., 12: 437.

[0100] Another form of an antibody fragment is a single domain antibody (dAb), sometimes referred to as a single chain antibody. Techniques for producing single domain antibodies are well known in the art (see, e.g., Cossins et al., Protein Expression and Purification, 2007, 51:253-59; Shuntao et al., Molec Immunol 06, 43 :1912-19; Tanha et al., J. Biol. Chem. 2001, 276: 24774-780). Other types of antibody fragments may comprise one or more complementarity determining regions (CDRs). CDR peptides ("minimal recognition units") can be obtained by constructing genes that encode the CDR of an antibody of interest. Said genes are prepared, e.g. eg, 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 Variations

[0101] In certain embodiments, the sequences of antibodies, such as the Fc portions of antibodies, can be varied to optimize physiological characteristics of the conjugates, such as half-life in serum. Methods of substituting amino acid sequences in proteins are well known in the art, such as site-directed mutagenesis (eg, Sambrook et al., Molecular Cloning, A Laboratory manual, 2nd edition, 1989). In preferred embodiments, the variation may involve the addition or removal of one or more glycosylation sites in the Fc sequence (eg, US Patent No. 6,254,868). In other preferred embodiments, specific amino acid substitutions can be made in the Fc sequence (eg, 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).

Antibody allotypes

[0102] The immunogenicity of therapeutic antibodies is associated with an increased risk of infusion reactions and decreased duration of therapeutic response (Baert et al, 2003, N Engl J Med 348:602-08). The degree to which therapeutic antibodies induce an immune response in the host may be determined in part by the allotype of the antibody (Stickler et al., 2011, Genes and Immunity 12:213-21). The antibody allotype is related to amino acid sequence variations at specific locations in the antibody constant region sequences. IgG antibody allotypes that contain a heavy chain y-type constant region are referred to as Gm allotypes (1976, J Immunol 117: 1056-59).

[0103] For common human IgG1 antibodies, the most prevalent allotype is G1m1 (Stickler et al, 2011, Genes and Immunity 12:213-21). However, the G1m3 allotype also occurs frequently in Caucasians (Stickler et al., 2011). G1m1 antibodies have been reported to contain allotypic sequences that tend to induce an immune response when administered to non-G1m1 (nG1m1) recipients, such as G1m3 patients (Stickler et al., 2011). The Antibodies of the non-Glml allotype are not as immunogenic when administered to G1 mi patients (Stickler et al., 2011).

[0104] The human G1 m1 allotype comprises the amino acid aspartic acid at Kabat position 356 and leucine at Kabat position 358 in the CH3 sequence of heavy chain IgG1. The nG1m1 allotype comprises the amino acid glutamic acid at Kabat position 356 and methionine at Kabat position 358. Both the G1 m1 and nG1 m1 allotypes comprise a glutamic acid residue at Kabat position 357 and the allotypes are sometimes referred to as DEL allotypes and EEM. A non-limiting example of the heavy chain constant region sequences for the G1m1 and nG1m1 allotype antibodies for the exemplary antibodies rituximab (SEQ ID NO: 85) and veltuzumab (SEQ ID NO: 86) is shown below.

Rituximab heavy chain variable region sequence ( SEQ ID NO: 85) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTH

TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES

NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

YTQKSLSLSPGK

Variable region heavy chain veltuzumab (SEQ ID NO: 86) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK

[0105] Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variations characteristic of IgG allotypes and their effect on immunogenicity. They reported that the G1m3 allotype is characterized by an arginine residue at Kabat position 214, compared to a lysine residue at Kabat 214 in the G1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acid at Kabat position 356, methionine at Kabat position 358, and alanine at Kabat position 431. The G1m1,2 allotype was characterized by aspartic acid at Kabat position 356, leucine at Kabat position Kabat 358 and glycine at Kabat position 431. In addition to sequence variants of the heavy chain constant region, Jefferis and Lefranc (2009) reported allotypic variants in the kappa light chain constant region, with the Km1 allotype characterized by valine at Kabat position 153 and leucine at Kabat position 191, the Km1,2 allotype by alanine at Kabat position 153 and leucine at Kabat position 191, and the Km3 allotype characterized by alanine at Kabat position 153 Kabat and Valine at Kabat position 191.

[0106] With respect to therapeutic antibodies, veltuzumab and rituximab are, respectively, humanized and chimeric IgG1 antibodies against CD20, of use for the therapy of a wide variety of hematologic malignancies and/or autoimmune diseases. Table 1 compares the allotype sequences of rituximab versus veltuzumab. As shown in Table 1, rituximab (G1m17.1) is a DEL IgG1 allotype, with an additional sequence variation at position 214 of Kabat (heavy chain CH1) from lysine in rituximab versus arginine in veltuzumab. Veltuzumab has been reported to be less immunogenic in subjects than rituximab ( see, eg, 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), an effect that has been attributed to the difference between humanized and chimeric antibodies. However, the difference in allotypes between the EEM and DEL allotypes probably also explains the lower immunogenicity of veltuzumab.

Table 1. Allotypes of Rituximab vs. Veltuzumab

[0107] In order to reduce the immunogenicity of therapeutic antibodies in individuals of the nG1m1 genotype, it is desirable to select the allotype of the antibody to correspond to the G1m3 allotype, characterized by arginine in Kabat 214, and the nG1m1,2 null-type allotype, characterized by glutamic acid at Kabat position 356, methionine at Kabat position 358, and alanine at Kabat position 431. 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 in common with the G1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356, methionine at Kabat 359, and alanine at Kabat 431. Since the immunogenicity appears to be related at least in part to the residues at At those locations, 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 may also be useful for therapeutic administration.

Known antibodies

[0108] In various embodiments of the invention as defined in the claims, any of a variety of antibodies known in the art may be used. Antibodies for use may be obtained commercially from a number of known sources. For example, a variety of antibody-secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA). A large number of antibodies against various disease targets, including but not limited to tumor associated antigens, have been deposited with the ATCC and/or variable region sequences have been published and are available for use in the claimed methods and compositions. See, eg, US Patent Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056 .509 7049060 7.045 132 7041 803 7041 .802 7041 .293 7.038018 7037 498 7 012 133 7.001 .598; 6 998, 468 6994976 6,994, 852 6989, 241 6,974, 863 6,965, 018 6964854 6962981 6 962813 6, 956 107 6 951 924 6949244 6 946 129 6943 020 6,939, 547 6921, 645 6921 645 6921 533 6919433 6 919, 078; 6,916,475 6905681 6,899,879 6893625 6,887,468 6,887466 6,884594 6881 405 6878812 6,875,580; 6,872 .568 6867006 6 864062 6 861 511 6861 .227 6861 226 6 .838282 6835549 6 835370 6 824 .780; 6 824 .778 6812206 6.793924 6 783 758 6.770 .450 6.767711 6764688 6764 681 6 764679 6 .743 .898; 6,733,981 6730307 6,720 155 6,716,966 6,709,653 6,693 176 6,692908 6689607 6,689,362 6,689,355; 6 682 .737 6682736 6.682 .734 6673 344 6.653104 6.652852 6 .635482 6630 144 6610 833 6610 .294; 6,605,441; 6,605,279 6,596,852; 6,592, 868; 6, 576, 745; 6,572, 856; 6,566,076; 6,562,618; 6.545 130 6544 .749; 6,534 .058 6528625 6.528 .269 6521 227 6518 .404 6511 .665 6 .491 915 6488930 6 482 598 6 .482 .408; 6,479 .247 6468531 6.468 .529 6465 173 6.461 .823 6.458356 6 .455044 6455040 6 451 310 6 .444 .206; 6 441 143 6432404 6.432 .402 6419 928 6413 726 6.406694 6 .403770 6403 091 6 395276 6 .395 .274; 6 387 .350 6383759 6.383 .484 6376 .654 6.372215 6.359 126 6 .355481 6355444 6 355245 6 .355 .244; 6 346, 246 6344 198 6,340, 571 6340, 459 6,331 175 6,306393 6, 254868 6187287 6 183744 6129914 6 120, 767 6096289 5,874, 540 5,900, 440 5,79856 5,736 ; 5,587,459; 5,443,953; 5,525,338. These are by way of example only and a wide variety of other antibodies and their hybridomas are known in the art. The skilled artisan will realize that antibody sequences or antibody-secreting hybridomas against almost any disease-associated antigen can be obtained by a simple search of the ATCC, NCBI, and/or USPTO databases for antibodies against a disease. target of interest associated with the selected disease. The antigen-binding domains of cloned antibodies can be amplified, excised, ligated into an expression vector, transfected into an adapted host cell, and used for protein production, using standard techniques well known in the art (see, e.g. ., US Patent Nos. 7,531,327, 7,537,930, 7,608,425 and 7,785,880).

[0109] Particular antibodies that may be of use in the present invention as defined in the claims include, but are not limited to LL 1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti -CD20), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti -epithelial glycoprotein-1 (EGP-1, also known as Tr Op -2)), pAm 4 or KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon specific p antigen), Immu 31 (an anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19 ), IMMU-H2B (anti-H2B), IMMU-H3 (anti-H3), IMMU-H4 (anti-H4), TAG-72 (eg, CC49), Tn, J591, or HuJ591 (anti-PSMA (antigen specific membrane membrane)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR ) german uzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20); PAM4 (also known as clivatuzumab, anti-mucin) and trastuzumab (anti-ErbB2). These antibodies are known in the art (eg, US patents UU US 5,686,072, 5,874,540, 6,107,090, 6,183,744, 6,306,393; 6,653,104, 6,730,300; 6,899,864,702; 7,074,403; 7,230,084; 7,238,785; 7,238,554 7,585,491; 7,612,180; 7,642,239; and US Patent Application Publication No. 20050271671; 20060193865; 20060210475; 20070087001; US Patent Application (US Patent No. 7,151,164), hA19 (US Patent No. 7,109,304), hIMMU-31 (US Patent No. 7,300,655), hLL1 ( US Patent No. 7,312,318), hLL2 (US Patent No. 5,789,554), hMu-9 (US Patent No. 7,387,772), hL243 (US Patent No. 7,612,180 ), hMN-14 (US Patent No. 6,676,924), hMN-15 (US Patent No. 8,287,865), hR1 (US Patent Application 13/688,812), hRS7 (Patent No. 7,238,785), hMN-3 (US Patent No. 7,541,440), AB-PG1-XG1-026 (US Patent Application 11/983,372, filed as ATCC PTA- 4405 and PTA-4406) and D2/B (WO 2009/130575).

[0110] Other useful antigens that can be targeted using the described conjugates include carbonic anhydrase IX, alpha-fetoprotein (AFP), α-actinin-4, A3, antibody-specific antigen A33, ART-4, B7, Ba 733, BAGE, Antigen BrE3, 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-1a, p-antigen colon-specific (CSAp), CEACAM5, CEACAM6, c-Met, DAM, EGFR, EGFRvIlI, EGP-1(TROP-2), EGP-2,ELF2-M, Ep-CAM, fibroblast growth factor (FGF ), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-p, histone H2B, histone H3, histone H4, HLA-DR, Hm 1.24, human chorionic gonadotropin (HCG), and their subs units, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, la, IGF-1R, IFN-y, IFN-a, IFN-p, IFN-A, 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, cell migration inhibitory factor macrophages (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, PD-L1, PD-1 receptor, growth factor placental, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC , TAG-72, tenascin, TRAIL receptors, TNF-a, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, EDB fibronectin, WT-1, 17-1A antigen, com complement C3, C3a, C3b, C5a, C5, a marker of angiogenesis, bcl-2, bcl-6, Kras, a marker oncogene, and an onc gene product (see, p. eg, 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).

[0111] A comprehensive analysis of suitable antigen (Cluster Designation, or CD) targets malignant hematopoietic cells, as shown by flow cytometry and may be a guide to selecting suitable antibodies for drug-conjugated immunotherapy, is Craig and Foon, Blood previously published online January 15, 2008; DOL 10.1182/blood-2007-11-120535.

[0112] CD66 antigens consist of five different glycoproteins with similar structures, CD66a-e, encoded by members of the carcinoembryonic antigen (CEA) gene family, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66 antigens (eg, CEACAM6) are expressed primarily on granulocytes, normal epithelial cells of the digestive tract, and tumor cells of various tissues. Also included as suitable targets for cancers are testicular cancer antigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007; 120 (11): 2411-7), as well as CD79a in myeloid leukemia (Kozlov et al., Cancer Genet. Cytogenet, 2005; 163 (1): 62-7) and also B-cell diseases, and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood 110 (2): 616-623 ). Several of the aforementioned antigens are described in US Provisional Application Serial No. 60/426,379, entitled "Use of Multi-specific, Non-covalent Complexes for Targeted Delivery of Therapeutics," filed November 15, 2002. Cancer stem cells, which are attributed to being populations of malignant precursor cells more resistant to therapy (Hill and Perris, J. Natl. Cancer Inst, 2007; 99: 1435-40), have antigens that can be targeted to 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 (Dalerba et al., Proc. Natl. Acad. Sci. USA 2007;104(24)10158-63), pancreatic cancer (Li et al., Cancer Res. 2007;67(3):1030-7), and in squamous cell carcinoma of head and neck (Prince et al., Proc. Natl. Acad. Sci. USA 2007; 104 (3) 973-8). Another useful target for breast cancer therapy is the LIV-1 antigen described by Taylor et al. (Biochem. J. 2003; 375: 51-9).

[0113] For multiple myeloma therapy, suitable targeting antibodies have been described for comparison, e.g. with CD38 and CD138 (Stevenson, MolMed2006;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).014*

[0114] Macrophage migration inhibitory factor (MIF) is an important regulator of innate and adaptive immunity and apoptosis. CD74 has been reported to be the endogenous receptor for MIF (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 for the treatment of a wide range of disease states, such as cancers of the bladder, prostate, breast, lung, colon, and chronic lymphocytic leukemia (p. eg, 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 c lupus erythematosus (Morand and Leech, 2005, Front Biosci 10: 12-22; Shachar and Haran, 2011, Leuk Lymphoma 52: 1446-54); renal 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 Mar 22, 2009; Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is an exemplary anti-CD74 antibody in therapeutic use for the treatment of diseases mediated by MIF.

[0115] Anti-TNF-α antibodies are known in the art and may be of use to treat immune diseases, such as autoimmune disease, immune dysfunction (eg, graft-versus-host disease, organ transplant rejection), or diabetes. Known antibodies against TNF-α include the human antibody CDP571 (Ofei et al., 2011, Diabetes 45: 881-85); murine MTNFAI, M2TNFAI, m3TNFAI, m3TNFABI, m302B, and m303 antibodies (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 invention as defined in the claims. Other antibodies of use for therapy of autoimmune or immune dysregulatory diseases include, but are not limited to anti-B cell antibodies such as veltuzumab, epratuzumab, milatuzumab or hL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25); daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3 receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-a4 integrin) and omalizumab (anti-IgE).

[0116] Checkpoint inhibitory antibodies have been used primarily in cancer therapy. Immune checkpoints refer to inhibitory pathways in the immune system that are responsible for maintaining self-tolerance and modulating the responsiveness of the immune system to minimize peripheral tissue damage. However, tumor cells can also activate immune system checkpoints to decrease the effectiveness of the immune response against tumor tissues. Exemplary checkpoint inhibitory antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152), programmed cell death protein 1 (PD1, also known as CD279), and programmed cell death ligand 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 pathogenic cells, tissues, or pathogens. Examples of anti-PD1 antibodies include lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), and pidilizumab (CT-011, CURETECH LTD.). Anti-PD1 antibodies are commercially available, e.g. from ABCAM® (AB137132), BIOLEGEND® (EH12,2H7, RMP1-14), and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH4). Exemplary 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). Examples of anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). Anti-PD1 antibodies are commercially available, e.g. 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 has recently received FDA approval for the treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11:89).

[0117] In another preferred embodiment, antibodies are used that are rapidly internalized and then re-expressed, processed, and displayed on cell surfaces, allowing continuous uptake and accumulation of circulating conjugate by the cell. An example of a more preferred antibody/antigen pair is LL1, an anti-CD74 MAb (invariant chain, specific chaperone class II, Ii) (see, eg, US Patent Nos. 6,653,104; 7,312,318). The CD74 antigen is highly expressed in B-cell lymphomas (including multiple myeloma) and leukemias, certain T-cell lymphomas, melanomas, colon, lung, and kidney cancers, glioblastomas, and some other cancers (Ong et al., Immunology 98: 296-302 (1999)). A review of the use of CD74 antibodies in cancer is found in Stein et al., Clin Cancer Res. 2007 Sep 15; 13 (18 Pt 2): 5556s-5563s.

[0118] Diseases that are preferably treated with anti-CD74 antibodies include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, melanoma, lung, kidney, colon cancers, glioblastoma multiforme, histiocytomas, myeloid leukemias, 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 reexpression of the antigen, allows the targeted LL1 antibody to be internalized along with any chemotherapeutic moieties it carries. This allows a high and therapeutic concentration of LL1 chemotherapeutic drug conjugate to accumulate within such cells. Internalized LL1 chemotherapeutic drug conjugates are cycled through lysosomes and endosomes, and the chemotherapeutic moiety is released in an active form into target cells.

[0119] Therapeutic conjugates can be used against pathogens, as antibodies against pathogens are known. For example, antibodies and antibody fragments that specifically bind to markers produced by or associated with infectious lesions, including viral, bacterial, fungal and parasitic infections, for example caused by pathogens such as bacteria, rickettsiae, mycoplasmas, protozoa, fungi and viruses, and antigens and products associated with such microorganisms have been described, among others, in Hansen et al., US Patent No. 3,927,193 and US Patent 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709 and 4,624,846, and in Reichert and Dewitz, cited above. A review listing antibodies against infectious organisms (antitoxin and antiviral antibodies), as well as other targets, is found in Casadevall, Clin Immunol 1999; 93 (1): 5-15012

[0120] Preferably, the pathogens are selected from the group consisting of HIV viruses, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin -resistant Staphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbis, lymphocytic choriomeningitis virus, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, reo viruses, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma bovisicum, Elbesmeria japonicum tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale , M. arginini, Acholeplasma laidlawii, M. salivarium, and M. pneumoniae, as described in US Patent No. 6,440,416.

[0121] In a more preferred embodiment, the drug conjugates of the present invention comprising anti-gp120 and other such anti-HIV antibodies can be used as therapeutic agents for HIV in AIDS patients; and Mycobacterium tuberculosis antibody drug conjugates are suitable as therapeutic agents for drug-refractory tuberculosis. Fusion proteins of anti-gp120 MAb (anti-HIV MAb) and a toxin, such as Pseudomonas exotoxin, have been examined for their antiviral properties (Van Oigen et al., J Drug Target, 5:75-91, 1998 ). Attempts to treat HIV infection in AIDS patients have failed, possibly due to insufficient efficacy or unacceptable host toxicity. The CPT drug conjugates of the present invention advantageously lack such toxic side effects of protein toxins and are therefore advantageously used in the treatment of HIV infection in AIDS patients. These drug conjugates can be administered alone or in combination with other antibiotics or therapeutic agents that are effective in such patients when administered alone. Candidate anti-HIV antibodies include the P4/D10 anti-envelope antibody described by Johansson et al. (AIDS. 2006 Oct 3; 20(15):1911-5), as well as anti-HIV antibodies disclosed and sold by Polymun (Vienna, Austria), also disclosed in US Patent 5,831,034, the US Patent 5,911,989 and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob. Chemother Agents. 2006; 50 (5): 1773-9. A preferred targeting agent for HIV is various combinations of these antibodies to overcome resistance.

[0122] Antibodies useful for treating autoimmune disease or disorders of the immune system (eg, graft-versus-host disease, organ transplant rejection) are known in the art and can be conjugated to CL2A-SN-38 using the described methods and compositions. Antibodies for use in treating autoimmune/immune dysfunction disease may bind to exemplary antigens including, but not limited to BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43, CD45, CD55, TNF-alpha, interferon, IL-6 and HLA-DR. Antibodies that bind to these and other target antigens, discussed above, can be used to treat autoimmune or immune dysfunction diseases. Autoimmune diseases that can be treated with immunoconjugates may include acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch purpura -Schonlein, poststreptococcal nephritis, erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, thromboangiitis obliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, bullous pemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, dorsal lateral scleroderosis salt, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, or fibrosing alveolitis.

[0123] The antibodies discussed above and other known antibodies against disease-associated antigens can be used as CL2A-SN-38 immunoconjugates, in the practice of the invention as defined in the claims.

Bispecific and multispecific antibodies

[0124] Bispecific antibodies are useful in a number of biomedical applications. For example, a bispecific antibody with binding sites for a tumor cell surface antigen and for a T cell surface receptor can direct the lysis of specific tumor cells by T cells. Bispecific antibodies that recognize gliomas and the CD3 epitope on T cells have been used successfully in the treatment of brain tumors in human patients (Nitta, et al. Lancet. 1990; 355: 368-371). In certain embodiments, the techniques and compositions for the conjugation of therapeutic agents described herein can be used with bispecific or multispecific antibodies as antibody moieties.0125

[0125] Numerous methods for producing bispecific or multispecific antibodies are known, as described, e.g.

eg, in US Patent No. 7,405,320. Bispecific antibodies can be produced by the quadroma method, which involves the fusion of two different hybridomas, each of which produces a monoclonal antibody that recognizes a different antigenic site (Milstein and Cuello, Nature, 1983; 305: 537-540).

[0126] Another method for producing bispecific antibodies uses heterobifunctional cross-linking 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 produced by reducing each of the two parent monoclonal antibodies to the respective half molecules, which are then mixed and allowed to reoxidize to obtain the hybrid structure (Staerz and Bevan. Proc Natl Acad Sci US A. 1986 ; 83:1453-1457). Another alternative involves chemically cross-linking two or three separately purified Fab' fragments using appropriate linkers. (See, eg, European Patent Application 0453082).

[0127] Other methods include improving the efficiency of generating hybrid hybridomas by gene transfer of different selectable markers via retrovirus-derived shuttle vectors into respective parental hybridomas, which are subsequently fused (DeMonte, et al. Proc Natl Acad Sci USA A. 1990, 87: 2941-2945); or transfection of a hybridoma cell line with expression plasmids containing the heavy and light chain genes of a different antibody.

[0128] The ^{cognate V h} and V ^l domains can be joined with a peptide linker of suitable composition and length (usually consisting of more than 12 amino acid residues). to form a single-chain Fv (scFv) with binding activity. Methods of making scFvs are described in US Patent No. 4,946,778 and US Patent No. 5,132,405. Reducing the length of the peptide linker to less than 12 amino acid residues prevents pairing of V ^h and V ^l domains on the same chain and forces pairing of V ^h and V ^l domains with complementary domains on other chains, which which results in the formation of functional multimers. Polypeptide chains domains V ^H and V ^L that bind with linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies). With linkers between 0 and 2 amino acid residues, trimers (referred to as triabodies) and tetramers (referred to as tetrabodies) are favored, but the exact patterns of oligomerization appear to depend on the composition and orientation of the V domains (linker V ^h - VL or V ^l -linker-VH), in addition to the length of the linker.

[0129] These techniques for producing multispecific or bispecific antibodies present various difficulties in terms of low yield, need for purification, low stability or the laboriousness of the technique. More recently, bispecific constructs known as "DOCK-AND-LOCK™" (DNL™) have been used to produce combinations of virtually any desired antibody, antibody fragment, and other effector molecules (see, eg, US Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and USSN 11/925,408). The technique uses complementary protein-binding domains, called anchor domains (EAs) and docking and dimerization domains (DDDs), which bind to each other and allow the assembly of complex structures, ranging from dimers, trimers, tetramers, quintamers and hexamers. These form stable complexes in high yield without the need for extensive purification. The technique allows the assembly of monospecific, bispecific, or multispecific antibodies. Any of the techniques known in the art for making bispecific or multispecific antibodies can be used in the practice of the methods now claimed.

[0130] Combinations of use, such as those preferred for cancer therapies, include CD20-CD22 antibodies, CD74-CD20 antibodies, Cd 74-CD22 antibodies, CEACAM5 (CEA) CEACAM6 (nCa) antibodies, growth factor-like insulin (ILGF) Antibodies CEACAM5, EGP-1(eg, RS-7) antibodies ILGF, CeAc AM5 Antibodies EGFR. Such antibodies need not only be used in combination, but can be combined as fusion proteins in a variety of ways, such as IgG, Fab, scFv, and the like, as described in US Patent Nos. 6,083,477; 6,183,744 and 6,962,702 and US Patent Application Publication Nos. 20030124058; 20030219433; 20040001825; 20040202666; 20040219156; 20040219203; 20040235065; 20050002945; 20050014207; 20050025709;20050079184; 20050169926; 20050175582; 20050249738; 20060014245 and 20060034759.

Pre-targeting

[0131] Bispecific or multispecific antibodies can also be used in pre-targeting techniques. Preselection is a multi-step process originally developed to resolve the slow blood clearance of directly targeted antibodies, which contributes to undesirable toxicity in normal tissues such as bone marrow. With preselection, a radionuclide or other therapeutic agent is attached to a small delivery molecule (targetable construct) that is cleared from the blood within minutes. First, a pretarget bispecific or multispecific antibody, which has binding sites for the target construct as well as a target antigen, is administered, free antibody is allowed to clear from the circulation, and then the target construct is administered. *

[0132] Preselection methods are described, e.g. eg, in Goodwin et al., US Patent No. 4,863,713; Goodwin et al., J. Nucl. Medicine. 29: 226, 1988; Hnatowich et al., J. Nucl. Medicine. 28: 1294, 1987; Oehr et al., J. Nucl. Medicine.

29: 728, 1988; Klibanov et al., J. Nucl. Medicine. 29: 1951, 1988; Sinitsyn et al., J. Nucl. Medicine. 30:66, 1989; Kalofonos et al., J. Nucl. Medicine. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991; Paganelli et al., Cancer Res. 51: 5960, 1991; Paganelli et al., Nucl. Medicine. Common. 12:211, 1991; US Patent No. 5,256,395; Stickney et al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51: 3119, 1991; US Patent No. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702; 7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and 6,962,702.

[0133] A screening method for treating or diagnosing a disease or disorder in a subject may be provided by: (1) administering to the subject a bispecific antibody or antibody fragment; (2) optionally administering to the subject a clearing composition and allowing the composition to remove the antibody from the circulation; and (3) administering to the subject the target construct, which contains one or more chemically bound or chelated therapeutic or diagnostic agents, such as SN-38.

target constructs

[0134] Target construct peptides labeled with one or more therapeutic or diagnostic agents for use in predissection may be selected to bind to a bispecific antibody with one or more binding sites for a target construct peptide and one or more sites binding agent for a target antigen associated with a disease or condition. Bispecific antibodies can be used in a prescreening technique where the antibody can first be administered to a subject. Sufficient time may be allowed for bispecific antibody to bind to a target antigen and for unbound antibody to clear from the circulation. A targetable construct, such as a labeled peptide, can then be administered to the subject and allowed to bind to the bispecific antibody and localize to the diseased cell or tissue.

[0135] Such targetable constructs can be diverse in structure and are selected not only for the availability of an antibody or fragment that binds with high affinity to the targeted construct, but also for rapid clearance in vivo when used within the target construct. pretargeting method and bispecific antibodies (bsAb) or multispecific antibodies. Hydrophobic agents are best for eliciting strong immune responses, while hydrophilic agents are preferred for rapid clearance in vivo. Thus, a balance is established between hydrophobic and hydrophilic character. This can be achieved, in part, by using hydrophilic chelating agents to compensate for the inherent hydrophobicity of many organic moieties. Furthermore, subunits of the addressable construct can be chosen that have opposite solution properties, e.g. eg, peptides, which contain amino acids, some of which are hydrophobic and others hydrophilic.

[0136] Peptides having as few as two amino acid residues, preferably two to ten residues, can be used and can also be coupled to other moieties, such as chelating agents. The linker should be a low molecular weight conjugate, preferably with a molecular weight of less than 50,000 daltons, and advantageously less than about 20,000 daltons, 10,000 daltons or 5,000 daltons. More usually, the addressable construct peptide will have four or more residues and one or more haptens to bind to, e.g. eg, to a bispecific antibody. Examples of haptens may include In-DTPA (indium diethylenetriaminepentaacetic acid) or h Sg (histamine succinylglycine). The target construct may also comprise one or more chelating moieties, such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4 ,7-triacetic), TETA (p-bromoacetamido-benzyltetraethylaminetetraacetic acid), NETA ([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino] acetic acid) or other known chelating moieties Chelating moieties may be used, eg, to bind a therapeutic or diagnostic radionuclide, paramagnetic ion, or the contrast agent.

[0137] The targeting construct may also comprise non-natural amino acids, e.g. eg, D-amino acids, in the backbone structure to increase peptide stability in vivo. In alternate embodiments, other backbone structures such as those constructed from non-natural amino acids or peptoids may be used.

[0138] Peptides used as targeting constructs are conveniently synthesized on an automated peptide synthesizer using a solid phase support and standard repetitive orthogonal deprotection and coupling techniques. Free amino groups on the peptide, which will be used later for conjugation of chelating moieties or other agents, are advantageously blocked with standard protecting groups such as a Boc group, while N-terminal residues can be acetylated to increase serum stability. . Said protecting groups are well known to those skilled in the art. See Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, n Y). When peptides are prepared for further use within the bispecific antibody system, they are advantageously cleaved from resins to generate the corresponding C-terminal amides, in order to inhibit carboxypeptidase activity in vivo.0139*

[0139] When preselection is used with bispecific antibodies, the antibody will contain a first binding site for an antigen produced by or associated with a target tissue and a second binding site for a hapten on the target construct. Examples of haptens include, but are not limited to HSG and In-DTPA. Antibodies raised against the HSG hapten are known (eg, antibody 679) and can be readily incorporated into the appropriate bispecific antibody (see, eg, US Patent Nos. 6,962,702; 7,138,103 and 7,300,644). However, other haptens and antibodies that bind to them are known in the art and can be used, such as In-DTPA and the 734 antibody (eg, US Patent No. 7,534,431).

DOCK-AND-LOCK™ (DNL™)

[0140] In preferred embodiments of the invention as defined in the claims, a bivalent or multivalent antibody is formed as a DOCK-AND-LOCK™ (DNL™) complex (see, eg, US Pat.

[0141] US Patent Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400). In general, the technique takes advantage of the high-affinity binding and specific interactions that occur between a cAMP-dependent protein kinase (PKA) regulatory subunit (R) dimerization and docking domain (DDD) sequence and a cAMP-dependent protein kinase (PKA) subunit. anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al., f Eb S Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5 : 959). Peptides DDD and dA can be attached to any protein, peptide, or other molecule. Because the DDD sequences spontaneously dimerize and bind to the DA sequence, the technique allows complex formation between any selected molecule that can bind to the DDD or DA sequences.

[0142] Although the standard DNL™ complex consists of a trimer with two DDD-linked molecules attached to one DA-linked molecule, variations in the complex structure allow for the formation of dimers, trimers, tetramers, pentamers, hexamers, and other multimers. In some embodiments, the DNL™ complex may comprise two or more antibodies, antibody fragments, or fusion proteins that bind to the same antigenic determinant or to two or more different antigens. The DNL™ complex may also comprise one or more effectors, such as proteins, peptides, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases such as onconase, inhibitory oligonucleotides such as siRNA, antigens or xenoantigens, polymers such as PEG, enzymes, therapeutic agents, hormones, cytotoxic agents, anti-angiogenic agents, proapoptotic agents, or any other molecule or aggregate.

[0143] PKA, which plays a central role in one of the best studied signal transduction pathways triggered by cAMP second messenger binding to R subunits, was first isolated from rabbit skeletal muscle in 1968 (Walsh et al ., J. Biol. Chem. 1968;243:3763). The holoenzyme structure consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989; 264: 8443). PKA isoenzymes are found with two types of R subunits (RI and RII), and each type has a and p isotherms (Scott, Pharmacol. Ther. 1991; 50: 123). Thus, the four isotherms of the PKA regulatory subunits are Ria, RIp, Rila, and RIIp. The R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues of Rila (Newlon et al., Nat. Struct. Biol. 1999; 6:222). As discussed below, similar portions of the amino acid sequences of other regulatory subunits are involved in dimerization and docking, each located near the N-terminus of the regulatory subunit. Binding of cAMP to R subunits leads to the release of catalytic subunits active for a broad spectrum of serine/threonine kinase activities, which are targeted to selected substrates through PKA compartmentalization via its coupling with AKAP (Scott et al., J. Biol. Chem. 1990;265;21561)

[0144] Since the first AKAP, microtubule-associated protein-2, was characterized in 1984 (Lohmann et al, Proc Natl. Acad Sci USA 1984;81:6723), more than 50 AKAPs that localize to various Subcellular sites, including the plasma membrane, actin cytoskeleton, nucleus, mitochondria, and endoplasmic reticulum, have been identified with diverse structures in species ranging from yeast to humans (Wong and Scott, Nat. Rev. Mol. Cell Biol 2004;5:959). The AKAP DA for PKA is an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.

1991; 266: 14188). The amino acid sequences of DA are quite varied among individual AKAps, with reported binding affinities for RII dimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA. 2003;100:4445). AKAPs will only bind to dimeric R subunits. For human Rila, DA binds to a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999; 6:216). Therefore, the dimerization domain and binding domain of human Rila AKAP are located within the same N-terminal 44 amino acid sequence (Newlon et al., Nat. Struct. Biol. 1999; 6: 222; Newlon et al., EMBO J. 2001;20:1651), referred to herein as DDD.0145

[0145] We have developed a platform technology to use the DDD of human PKA regulatory subunits and the DA of AKAP as an excellent pair of linker modules for the coupling of any two entities, hereafter referred to as A and B, in a complex non-covalent, which could be further locked into a DNL™ complex by introducing cysteine residues in both DDD and DA at strategic positions to facilitate disulfide bond formation. The general methodology of the approach is as follows. Entity A is constructed by joining a DDD sequence to a precursor of A , resulting in a first component referred to hereinafter as a . Because the DDD sequence would affect the spontaneous formation of a dimer, A would be composed of a 2 . Entity B is constructed by joining a sequence of DA to a precursor of B , resulting in a second component referred to hereinafter as b. The DDD dimeric motif contained in a 2 will create a docking site to bind to the DA sequence contained in b, thus facilitating easy association of a 2 and b to form a binary trimeric complex composed of a 2 b. This binding event becomes irreversible with a subsequent reaction to covalently secure the two entities through disulfide bonds, which occurs very efficiently according to the effective local concentration principle because the initial binding interactions must carry the reactive thiol groups. placed in both the DDD and the DA. in proximity (Chmura et al., Proc. Natl. Acad. Sci. USA 2001;98:8480) to bind specifically the site. Using various combinations of linkers, adapter modules, and precursors, a wide variety of DNL™ constructs of different stoichiometry can be produced and used (see, e.g., US Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400).

[0146] By connecting DDD and DA away from the functional groups of the two precursors, such site-specific ligations are also expected to preserve the original activities of the two precursors. This approach is modular in nature and can potentially be applied to covalently and site-specifically bind a wide range of substances, including peptides, proteins, antibodies, antibody fragments, and other effector moieties with a wide range of activities. Using the fusion protein method for constructing DA and DDD-conjugated effectors described in the Examples below, virtually any protein or peptide can be incorporated into a DNL™ construct. However, the technique is not limiting and other methods of conjugation can be used.

[0147] A variety of methods are known for making fusion proteins, including nucleic acid synthesis, hybridization, and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest. Such double-stranded nucleic acids can be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, eg, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, 1989). . In such preferred embodiments, the DA and/or DDD moiety can be attached to the N-terminal or C-terminal end of an effector protein or peptide. However, one skilled in the art will appreciate that the binding site of a DA or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the moiety. effector involved in its physiological activity. Site-specific binding of a variety of effector moieties can be accomplished using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.

Structure-function relationships in DA and DDD residues

[0148] For different types of DNL™ constructs, different DA or DDD sequences can be used. Exemplary sequences of DDD and DA are provided below.

DDD1

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:1)

DDD2

CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 2)

AD1

QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)

AD2

CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO: 4)

[0149] The skilled artisan will realize that DDD1 and DDD2 are based on the DDD sequence of the human Rila isoform of protein kinase A. However, in alternative embodiments, the DDD and DA residues may be based on the sequence DDD of the human Rla form of protein kinase A and a corresponding AKAp sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3

SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK (SEQ ID NO: 5)

DDD3C

MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEE

AK (SEQ ID NO:6)

AD3

CGFEELAWKIAKMIWSDVFQQGC (SEQ ID NO: 7).

[0150] In other alternate embodiments, other DA sequence variants and/or DDL™ complexes may be used in the construction of DND™ moieties. For example, there are only four human PKA DDD sequence variants, corresponding to the PKA DDD residues Rla, Rila, Plp, and Rllp. The Rila DDD sequence is the basis of DDD1 and DDD2 described above. The four human PKA DDD sequences are shown below. The sequence DDD represents residues 1-44 of Rila, 1-44 of Rllp, 12-61 of Rla and 13-66 of Rlp. (Note that the sequence of DDD1 is slightly modified from the human PKA Rlla DDD moiety.)

PKA Rla

SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK (SEQ ID NO: 8)

PKA Rlp

SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA (SEQ ID NO: 9)

PKA Rlla

SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ (SEQ ID NO: 10)

PKA RII/3

SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER (SEQ ID NO: 11)

[0151] The structure-function relationships of the DA and DDD domains have been the subject of investigation. (See, e.g., Burns-Hamuro et al., 2005, Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA. 100: 4445-50; Hundsrucker et al., 2006, Biochem J 396: 297-306; Stokka et al., 2006, Biochem J 400: 493-99; Gold et al., 2006, Mol Cell 24: 383-95.; Kinderman et al, 2006, Mol Cell 24: 397-408).

[0152] For example, Kinderman et al. (2006, Mol Cell 24: 397-408) examined the crystal structure of the DA-DDD binding interaction and concluded that the human DDD sequence contained several conserved amino acid residues that were important in dimer formation or DDD binding. AKAP, underlined in SEQ ID NO: 1 below. (See Figure 1 of Kinderman et al., 2006.) One skilled in the art will appreciate that in designing sequence variants of the DDD sequence, one would desirably avoid changing any of the underlined residues, while substitutions could be made. conservative amino acids for residues that are less critical for AKAP dimerization and binding.

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:1)

[0153] As discussed in more detail below, conservative amino acid substitutions have been characterized for each of the twenty common L-amino acids. Therefore, based on data from Kinderman (2006) and conservative amino acid substitutions, potential alternative DDD sequences based on SEQ ID NO: 1 are shown in Table 2. In designing Table 2, only substitutions of highly conservative amino acids. For example, charged residues were only replaced by residues of the same charge, residues with small side chains were replaced by residues of similar size, hydroxyl side chains were only substituted by other hydroxyls, etc. Due to the unique effect of proline on amino acid secondary structure, no other residues were substituted for proline. A limited number of such potential alternative DDD residue sequences are shown in SEQ ID NO: 12 through SEQ ID NO: 31 below. The skilled artisan will realize that a large number of alternative species within the genus of DDD moieties can be constructed by standard techniques, e.g. eg, using a commercial peptide synthesizer or well-known site-directed mutagenesis techniques. The effect of amino acid substitutions on DA moiety binding can also be readily determined by standard binding assays, e.g. eg, as described in Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50).

Table 2. Conservative amino acid substitutions in DDD1 (SEQ ID NO: 1). Consensus sequence described as SEQ ID NO: 87.

THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 12) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13) SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14) SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15) SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16) SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17) SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA ( SEQ ID NO: 18) SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19) SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20) SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21) SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22) SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23) SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24) SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25) SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26) SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27) SHIQIPPGLTELLQGYTVEVLRQQ PPDLVEFLVEYFTRLREARA (SEQ ID NO: 28) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)

[0154] Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed a bioinformatic analysis of the DA sequence of several AKAP proteins to design an RII-selective DA sequence named AKAP-IS (SEQ ID NO: 3), with a binding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence where substitutions tended to decrease DDD binding are underlined in SEQ ID NO: 3 below. One skilled in the art will appreciate that when designing sequence variants of the DA sequence, one would desirably avoid changing any of the underlined residues, while conservative amino acid substitutions could be made for residues that are less critical for DDD binding. . Table 3 shows possible conservative amino acid substitutions in the sequence of AKAP-IS (AD1, SEQ ID NO: 3), similar to that shown for DDD1 (SEQ ID NO:1) in Table 2 above.

[0155] A limited number of such potential alternative DA residue sequences are shown in SEQ ID NO: 32 to SEQ ID NO: 49 below. Again, one skilled in the art could prepare, test and use a large number of species within the genus of possible DA residue sequences, based on the data of Alto et al. (2003). It is noted that Figure 2 from Alto (2003) shows an even greater number of possible amino acid substitutions that can be made, while retaining binding activity to DDD residues, based on actual binding experiments. AKAP-IS

QIEYLAKQJVDNAIQQA (SEQ ID NO: 3)

Table 3. Conservative amino acid substitutions in AD1 (SEQ ID NO: 3). Consensus sequence described as SEQ ID NO: 88.

NIEYLAKQIVDNAIQQA (SEQ ID NO: 32)

QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)

QVEYLAKQIVDNAIQQA (SEQ ID NO: 34)

QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)

QIEFLAKQIVDNAIQQA (SEQ ID NO: 36)

QIETLAKQIVDNAIQQA (SEQ ID NO: 37)

QIESLAKQIVDNAIQQA (SEQ ID NO: 38)

QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)

QIEYVAKQIVDNAIQQA (SEQ ID NO: 40)

QIEYLARQIVDNAIQQA (SEQ ID NO: 41)

QIEYLAKNIVDNAIQQA (SEQ ID NO: 42)

QIEYLAKQIVENAIQQA (SEQ ID NO: 43)

QIEYLAKQIVDQAIQQA (SEQ ID NO: 44)

QIEYLAKQIVDNAINQA (SEQ ID NO: 45)

QIEYLAKQIVDNAIQNA (SEQ ID NO: 46)

QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)

QIEYLAKQIVDNAIQQI (SEQ ID NO: 48)

QIEYLAKQIVDNAIQQV (SEQ ID NO: 49)

[0156] Gold et al. (2006, Mol Cell 24: 383-95) used crystallography and peptide selection to develop a SuperAKAPIS sequence (SEQ ID NO: 50), which exhibits five orders of magnitude higher selectivity for the RII isoform of PKA compared to the isoform RI. Underlined residues indicate the positions of amino acid substitutions, relative to the AKAP-IS sequence, that increased binding to the DDD residue of Rila. In this sequence, the N-terminal Q residue is numbered as residue number 4 and the C-terminal A residue is residue number 20. Residues at which substitutions could be made to affect Rila affinity were residues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It is contemplated that in certain alternate embodiments, the SuperAKAP-IS sequence may be substituted for the AKAP-IS DA moiety sequence to prepare DNL™ constructs. Other alternative sequences that could be substituted for the AKAP-IS DA sequence are shown in SEQ ID NO: 51-53. Substitutions relative to the AKAP-IS sequence are underlined. It is anticipated that, as with the AD2 sequence shown in SEQ ID NO: 4, the DA moiety may also include additional N-terminal cysteine and glycine residues and C-terminal glycine and cysteine residues.

SuperAKAP-IS

QIEYVAKQIVDYAIHQA (SEQ ID NO: 50)

Alternative AKAP sequences

QIEYKAKQIVDHAIHQA (SEQ ID NO: 51)

QIEYHAKQIVDHAIHQA (SEQ ID NO: 52)

QIEYVAKQIVDHAIHQA (SEQ ID NO: 53)

[0157] Figure 2 of Gold et al. revealed additional DDD-binding sequences from a variety of AKAP proteins, shown below.

RII-specific AKAPs

[0158]

AKAP-KL

PLEYQAGLLVQNAIQQAI (SEQ ID NO: 54)

AKAP79

LLIETASSLVKNAIQLSI (SEQ ID NO: 55)

AKAP-Lbc

LIEEAASRIVDAVIEQVK (SEQ ID NO: 56)

IR-specific AKAPs

[0159]

AKAPce

ALYQFADRFSELVISEAL (SEQ ID NO: 57)

RIAD LEQVANQLADQIIKEAT (SEQ ID NO: 58)

SS38

FEELAWKIAKMIWSDVF (SEQ ID NO: 59)

Dual specificity AKAPs

[0160]

AKAP7

ELVRLSKRLVENAVLKAV (SEQ ID NO: 60)

MAP2D

TAEEVSARIVQVVTAEAV (SEQ ID NO: 61)

DAKAP1

QIKQAAFQLISQVILEAT (SEQ ID NO: 62)

DAKAP2

LAWKIAKMIVSDVMQQ (SEQ ID NO: 63)

[0161] Stokka et al. (2006, Biochem J 400: 493-99) also developed competitor peptides of AKAP binding to PKA, shown in Se Q ID NO: 64-66. Peptide antagonists were designated Ht31 (SEQ ID NO: 64), RIAD (SEQ ID NO: 65), and PV-38 (SEQ ID NO: 66). Peptide Ht-31 exhibited a higher affinity for the RII isoform of PKA, while RIAD and PV-38 showed higher affinity for RI.

Ht31

DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 64)

RIAD LEQYANQLADQIIKEATE (SEQ ID NO: 65)

PV-38

FEELAWKIAKMIWSDVFQQC (SEQ ID NO: 66) 0162*

[0162] Hundsrucker et al. (2006, Biochem J 396:297-306) developed yet other competitor peptides for binding of AKAP to PKA, with a binding constant as low as 0.4 nM to the DDD of the RII form of PKA. The sequences of various AKAP antagonist peptides are provided in Table 1 of Hundsrucker et al., reproduced in Table 4 below. AKAPIS represents a synthetic peptide that binds to the RII subunit. All other peptides are derived from the RII binding domains of the indicated AKAPs.

Table 4. AKAP peptide sequences

Peptide Sequence____________________________

AKAPIS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)

AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO: 67)

Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68)

Ht31-P KGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69)

AKAP75-wt-pep PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70)

AKAP75-L304T-pep PEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO:71)

AKAP75-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72)

AKAP75-P-pep PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73)

AKAP75-PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74)

AKAP75-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75)

AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76)

AKAP2-pep LVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77)

AKAP5-pep QYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78)

AKAP9-pep LEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79)

AKAP10-pep NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80)

AKAP11-pep VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81)

AKAP12-pep NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82)

AKAP14-pep TQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83)

Rab32-pep ETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

[0163] Residues that were highly conserved between the DA domains of different AKAP proteins are indicated below underlining with reference to the AKAP IS sequence (SEQ ID NO: 3). The residues are the same as those observed by Alto et al. (2003), with the addition of the C-terminal alanine residue. (See Figure 4 of Hundsrucker et al. (2006).) Peptide antagonist sequences with particularly high affinities for the RII DDD sequence were AKAP-IS, AKAP75-wtpep, AKAP75-L304T-pep, and AKAP75-L308D -pep.

AKAP-IS

QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)

[0164] Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree of sequence homology between different AKAP-binding DDD sequences of human and non-human proteins and identified residues in the DDD sequences that appeared to be the most conserved between different DDD halves. . These are indicated below underlining with reference to the human Rila DDD PKA sequence of SEQ ID NO: 1. Residues that were especially conserved are indicated in italics. The residues overlap, but are not identical to those suggested by Kinderman et al. (2006) is important for binding to AKAP proteins. The skilled artisan will realize that when designing sequence variants of DDD, it would be more preferable to avoid changing the most conserved residues (in italics), and it would also be preferable to avoid changing conserved residues (underlined), while substitutions of Conservative amino acids can be considered for residues that are not underlined or italicized.

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:1)

[0165] A modified set of conservative amino acid substitutions for the DDD1 sequence (SEQ ID NO: 1), based on data from Carr et al. (2001) is shown in Table 5. Alternative DDD amino acid sequences could easily be derived by the skilled person as described above for Table 1 and Table 2.

Table 5. Conservative amino acid substitutions in DDD1 (SEQ ID NO: 1). Consensus sequence described as SEQ ID NO: 89016*

[0166] One of skill in the art will appreciate that these and other amino acid substitutions in the DDD or DA amino acid sequences can be used to produce alternative species within the genus of DA or DDD residues, using techniques that are standard in the art. and only routine experimentation.

DNL™ Alternative Structures

[0167] In certain alternative embodiments, DNL™ constructs may be formed using alternatively constructed antibodies or antibody fragments, in which a DA moiety may be attached at the C-terminus of the kappa light chain (C k ), instead of the C-terminus of the Fc on the heavy chain. Alternative formed DNL™ constructs can be prepared as described in US Provisional Patent Application Serial Numbers 61/654,310, filed June 1, 2012, 61/662,086, filed June 20, 2012, 61 / 673.553, filed 19 July 2012 and 61 / 682.531, filed August 13, 2012. the DNL constructs ™ conjugated light chain exhibit improved effector function activity in vitro and Fc pharmacokinetics, stability and activity improved antilymphoma in vivo (Rossi et al., 2013, Bioconjug Chem 24: 63-71).

[0168] ^{C k} -conjugated DNL™ constructs can be prepared as described in US Provisional Patent Application ^{Serial Nos.} 61/654,310, 61/662,086, 61/673,553, and 61/682,531. Briefly, C ^k -AD2-IgG, was generated by recombinant engineering, whereby the AD2 peptide was fused to the C-terminus of the kappa light chain. Because the natural C-terminus of C ^k is a cysteine residue, which forms a disulfide bridge to C ^h 1 , a 16-amino acid residue "hinge" linker was used to space AD2 from the C ^k -V disulfide bridge. ^h 1. Mammalian expression vectors for C ^k -AD2-IgG-veltuzumab and C ^k -AD2-IgG-epratuzumab were constructed using the pdHL2 vector, which was previously used for expression of homologous modules of C ^H 3 - AD2-IgG. A 2208 bp nucleotide sequence was synthesized comprising the pdHL2 vector sequence running from the Bam HI restriction site within the V ^k /C ^k intron to the Xho I 3' restriction site of ^{the C k} intron, with the insertion of the coding sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ ID NO: 162) and AD2, in frame at the 3' end of the coding sequence for CK. This synthetic sequence was inserted into the IgG-pdHL2 expression vectors for veltuzumab and epratuzumab through the Bam HI and Xho I restriction sites. Generation of production clones with SpESFX-10 was performed as described for modules C ^H 3-AD2-IgG. C ^k -AD2-IgG-veltuzumab and C ^k -AD2-IgG-epratuzumab were produced by stably transfected production clones in batch roller bottle culture and purified from the supernatant in a single step using protein affinity chromatography. To MabSelect (GE Healthcare).

[0169] Following the same DNL™ process as previously described for 22-(20)-(20) (Rossi et al, 2009, Blood. 113:6161-71), C ^k -AD2-IgG-epratuzumab was conjugated with C ^H 1-DDD2-Fab-veltuzumab, a Fab-based module derived from veltuzumab, to generate bsHexAb 22*-(20)-(20), where the 22* indicates the C ^k -AD2 module of epratuzumab and each (20) symbolizes a stabilized dimer of veltuzumab Fab. The properties of 22 * - (20) - (20) were compared with those of 22- (20) - (20), the Fc-bsHexAb homologue comprising C ^H 3-AD2-IgG-epratuzumab, having a composition and Similar molecular structure, but a different architecture.

[0170] Following the same DNL™ process previously described for 20-2b (Rossi et al, 2009, Blood 114: 3864-71), C ^k -AD2-IgG-veltuzumab, was conjugated with IFNa2b-DDD2, a module of IFNa2b with a DDD2 peptide fused at its C-terminus, to generate 20*-2b, comprising veltuzumab with a dimeric IFNa2b fused to each light chain. The properties of 20*-2b were compared with those of 20-2b, which is the Fc-IgG-IFNa homologue.

[0171] Each of the bsHexAbs and IgG-IFNa were isolated from the DNL™ reaction mixture by MabSelect affinity chromatography. The two C ^k -derived prototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprising epratuzumab (anti-CD22) and four veltuzumab (anti-CD20) Fabs, and a CD20-targeted immunocytokine, comprising veltuzumab and four anti-CD20 molecules. of interferon-a2b, showed improved Fc effector functions in vitro, as well as improved pharmacokinetics, stability, and anti-lymphoma activity in vivo, compared to its Fc-derived counterparts.

amino acid substitutions

[0172] In alternative embodiments, the disclosed methods and compositions may involve the production and use of proteins or peptides with one or more substituted amino acid residues. For example, the DDD and/or dA sequences used to make DNL™ constructs can be modified as discussed above.

[0173] One skilled in the art will be aware that, in general, amino acid substitutions typically involve the substitution of one amino acid with another amino acid of relatively similar properties (ie, conservative amino acid substitutions). The properties of various amino acids and the effect of amino acid substitution on protein structure and function have been the subject of extensive study and skill in the art.0174*

[0174] For example, the hydropathic index of amino acids can be considered (Kyte and Doolittle, 1982, J. Mol Biol. 157: 105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the protein's interaction with other molecules. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). When making conservative substitutions, the preferred use of amino acids whose hydropathic indices are within 62, are more preferred within ± 1 and are even more preferred within ± 0.5.

[0175] Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (eg, US Patent No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 .+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Substitution of amino acids with others of similar hydrophilicity is preferred.

[0176] Other considerations include the size of the amino acid side chain. For example, it would generally not be preferable to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g. eg, tryptophan or tyrosine. The effect of various amino acid residues on the secondary structure of the protein is also a consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha helix, beta sheet, or reverse turn secondary structure has been determined and is known in the art ( see, eg, Chou & Fasman, 1974 Biochemistry 13:222-245, 1978 Ann Rev Biochem 47:251-276, 1979 Biophys J 26:367-384).

[0177] Based on such considerations and extensive empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. For example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) wing, be; Gln(Q) glu, asn; Glu (E) gln, asp; Gly (G) wing; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg; Met(M)phe,ile,leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) wing; Be (S), thr; Thr(T)be; Trp(W)phe,tyr; Tyr(Y)trp, phe, thr, ser; Val (V)ile, leu, met, phe, ala.

[0178] Other considerations for amino acid substitutions include whether or not the residue is within a protein or exposed to solvent. For interior residues, conservative substitutions would include: Asp and Asn; Ser and Thr; Being and Wing; Thr and Ala; Wing and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp. (See, eg, the PROWL website at rockefeller.edu) For solvent-exposed residues, conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Wing and Pro; Wing and Gly; Wing and Being; Wing and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Several matrices have been constructed to aid in the selection of amino acid substitutions, such as the PAM250 scoring matrix, the Dayhoff matrix, the Grantham matrix, the McLachlan matrix, the Doolittle matrix, the Henikoff matrix, the Miyata matrix, the Fitch matrix, the Jones matrix, the Rao matrix, the Levin matrix and the Risler matrix ( Idem.)

[0179] In determining amino acid substitutions, one can also consider the existence of intermolecular or intramolecular bonds, such as ionic bond formation (salt bridges) between positively charged residues (eg, Su, Arg, Lys ) and negatively charged residues (eg, Asp, Glu) or disulfide bonds between nearby cysteine residues.

[0180] Methods for substituting any amino acid for any other amino acid in an encoded protein sequence are well known and a matter of routine experimentation for those skilled in the art, e.g. eg, by the technique of site-directed mutagenesis or by oligonucleotide synthesis and assembly. encoding an amino acid substitution and splicing into an expression vector construct.

Phage display

[0181] Certain embodiments of the invention as defined in the claims may relate to peptide binding and/or peptide mimetics of various target molecules, cells or tissues. Binding peptides can be identified by any method known in the art, including but not limited to the phage display technique. Various phage display methods and techniques for producing various populations of peptides are well known in the art. For example, US Patent No. 5,223,409; 5,622,699 and 6,068,829 describe methods for preparing a phage library. The phage display technique involves the genetic manipulation of bacteriophages so that small peptides can be displayed on their surface (Smith and Scott, 1985, Science 228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. 21:228-257 ). In addition to peptides, larger protein domains, such as single-chain antibodies, can also be displayed on the surface of phage particles (Arap et al., 1998, Science 279: 377-380).0182*

[0182] Selective target amino acid sequences for a given organ, tissue, cell type, or target molecule can be isolated by washing (Pasqualini and Ruoslahti, 1996, Nature 380: 364-366; Pasqualini, 1999, The Quart J. Nucl. Med. 43:159-162). Briefly, a library of phage containing putative targeting peptides is administered to an intact organism or to isolated organs, tissues, cell types, or target molecules, and samples containing bound phage are collected. Phage that bind to a target can be eluted from a target organ, tissue, cell type, or target molecule and then amplified by growing them in bacterial hosts.

[0183] In certain examples, the phage can be propagated in host bacteria between rounds of washing. Instead of being lysed by the phage, the bacteria can secrete multiple copies of the phage displaying a particular insert. If desired, the amplified phage can be exposed to the target organs, tissues, cell types, or target molecule again and harvested for additional rounds of panning. Multiple rounds of screening can be performed until a population of selective or specific binders is obtained. The amino acid sequence of the peptides can be determined by sequencing the DNA corresponding to the targeting peptide insert in the phage genome. The identified targeting peptide can then be produced as a synthetic peptide by standard protein chemistry techniques (Arap et al., 1998, Smith et al., 1985).

[0184] In some examples, a subtraction protocol can be used to further reduce background phage binding. The purpose of subtraction is to remove phages from the library that bind to targets other than the target of interest. In alternative embodiments, the phage library may be preselected against a control cell, tissue, or organ. For example, tumor-binding peptides can be identified after prescreening a library against a control normal cell line. After subtraction, the library can be screened against the molecule, cell, tissue, or organ of interest. Other methods of subtraction protocols are known and can be used in the practice of the claimed methods, e.g. eg, as described in US Patent Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

nanobodies

[0185] Nanobodies are single domain antibodies of about 12-15 kDa in size (about 110 amino acids in length). Nanobodies can selectively bind target antigens, like full-size antibodies, and have similar affinities for antigens. However, due to their much smaller size, they may be able to better penetrate solid tumors. The smaller size also contributes to the stability of the nanobody, which is more resistant to extremes of pH and temperature than full-size antibodies (Van Der Linden et al., 1999, Biochim Biophys Act 1431: 37-46). Single domain antibodies were originally developed following the discovery that camelids (camels, alpacas, llamas) possess fully functional antibodies without light chains (e.g., Hamsen et al., 2007, Appl Microbiol Biotechnol 77: 13-22 ). Heavy chain antibodies consist of a single variable domain (V ^hh ) and two constant domains (C ^h 2 and C ^h 3). Like antibodies, nanobodies can be developed and used as multivalent and/or bispecific constructs. Humanized forms of nanobodies that target a variety of target antigens, including IL-6R, vWF, TNF, RSV, RANKL, IL-17A&F, and IgE (eg, ABLy Nx®, Ghent, Belgium), with potential clinical use in cancer, inflammation, infectious diseases, Alzheimer's disease, acute coronary syndrome, and other disorders (eg, Saerens et al., 2008, Curr Opin Pharmacol 8: 600-8; Muyldermans, 2013 , Ann Rev Biochem 82:775-97; Ibanez et al., 2011, J Infect Dis 203:1063-72).

[0186] The plasma half-life of nanobodies is shorter than that of full-size antibodies, with elimination primarily via the kidneys. Because they lack an Fc region, they do not exhibit complement-dependent cytotoxicity.

[0187] Nanobodies can be produced by immunizing camels, llamas, alpacas, or sharks with the target antigen, followed by isolation of mRNA, cloning in libraries, and screening for antigen binding. Nanobody sequences can be humanized by standard techniques (eg, Jones et al., 1986, Nature 321:522, Riechmann et al., 1988, Nature 332:323, Verhoeyen et al., 1988, Science 239: 1534, Carter et al., 1992, Proc Nat'l Acad Sci USA 89:4285, Sandhu, 1992, Crit Rev Biotech 12:437, Singer et al., 1993, J Immun 150: 2844). Humanization is relatively straightforward due to the high homology between camelid and human FR sequences.0189*

[0188] In various embodiments, the subject CL2A-SN-38 conjugates may comprise nanobodies for targeted delivery of conjugated drug to cells, tissues, organs, or pathogens. Nanobodies of use are described, e.g. eg, in US Patent Nos. 7,807,162; 7,939,277; 8,188,223; 8,217,140; 8,372,398; 8,557,965; 8,623,361 and 8,629,244.)

conjugation protocols

[0189] The preferred conjugation protocol is based on thiol-maleimide, a thiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamide reaction which are easy at neutral or acidic pH. This avoids the need for higher pH conditions for conjugations, e.g. For example, they would be necessary when active esters are used. More details of exemplary conjugation protocols are described below in the Examples section.

therapeutic treatment

[0190] In another aspect, the present disclosure relates to a method of treating a subject, comprising administering a therapeutically effective amount of a therapeutic conjugate as described herein to a subject. Diseases that can be treated with the therapeutic conjugates described herein include, but are not limited to, B-cell malignancies (e.g., non-Hodgkin's lymphoma, mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma, diffuse non-Hodgkin's lymphoma, B large, Burkitt's lymphoma, follicular lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia) using e.g. g., an anti-CD22 antibody such as the hLL2 MAb (epratuzumab, see US Patent No. 6,183,744), against another CD22 epitope (hRFB4), or antibodies against other B-cell antigens, 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 epithelia of the digestive system of endodermal origin, 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, e.g. eg, an oncofetal antigen, produced by or associated with a malignant solid tumor or hematopoietic neoplasm, e.g. eg, a gastrointestinal tract, stomach, colon, esophagus, a liver, lung, breast, pancreas, liver, prostate, ovary, testicular, brain, bone or lymphatic tumor, a sarcoma or a melanoma are advantageously used. Such therapies may be administered once or repeatedly, depending on the disease state and tolerability of the conjugate, and may also optionally be used in combination with other therapeutic modalities, such as surgery, external beam radiation, radioimmunotherapy, immunotherapy, chemotherapy, antisense therapy, RNA interference therapy, gene therapy and the like. Each combination will be tailored to tumor type, stage, patient status and prior therapy, and other factors considered by the responsible physician.

[0191] As used herein, the term "subject" refers to any animal (ie, vertebrates and invertebrates), including, but not limited to mammals, including humans. The term is not intended to be limited to any particular age or gender. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are encompassed by the term. The doses given in this document are for humans, but can be adjusted to the size of other mammals, as well as children, according to weight or size per square meter.

[0192] In a preferred example, therapeutic conjugates comprising an anti-EGP-1 (anti-TROP-2) antibody such as the hRS7 MAb can be used to treat carcinomas such as carcinomas of the esophagus, pancreas, lung, stomach, colon and rectum, urinary bladder, breast, ovary, uterus, kidney, and prostate, as described in US Patent No. 7,238,785; 7,517,964 and 8,084,583. An antibody against hRS7 is a humanized antibody comprising CDR1 light chain complementarity determining region (CDR) sequences (KASQDVSIAVA, SEQ ID NO: 90); CDR2 (SASYRYT, SEQ ID NO: 91); and CDR3 (QQHYITPLT, SEQ ID NO: 92) and heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO: 93); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO: 94) and CDR3 (GGFGSSYWYFDV, SEQ ID NO: 95)

[0193] In another example, therapeutic conjugates comprising an anti-CEACAM5 antibody (eg, hMN-14, labretuzumab) and/or an anti-CEACAM6 antibody (eg, hMN-3 or hMN-15 ) can be used to treat any of a variety of cancers that express CEACAM5 and/or CEACAM6, as described in US Patent Nos. 7,541,440; 7,951,369; 5,874,540; 6,676,924 and 8,267,865. Solid tumors that can be treated with anti-CEACAM5, anti-CEACAM6, or a combination of the two include, but are not limited to, breast, lung, pancreas, esophagus, medullary thyroid, ovary, colon, rectum, urinary bladder, mouth, and uterine cancers. stomach. Most carcinomas, including gastrointestinal, respiratory, genitourinary, and breast cancers, express CEACAM5 and can be treated with the immunoconjugates in question. An hMN-14 antibody is a humanized antibody comprising the light chain variable region CDR sequences CDR1 (KASQDVGTSVA; SEQ ID NO: 96), CDR2 (WTSTRHT; SEQ ID NO: 97) and CDR3 (QQYSLYRS; SEQ ID NO: 98), and the heavy chain variable region CDR sequences CDR1 (TYWMS; SEQ ID NO: 99), CDR2 (EIHPDSSTINYAPSLKD; SEQ ID NO: 100) and CDR3 (LYFGFPWFAY; SEQ ID NO: 101).

[0194] An hMN-3 antibody is a humanized antibody comprising the light chain variable region CDR sequences CDR1 (RSSQSIVHSNGNTYLE, SEQ ID NO: 102), CDR2 (KVSNRFS, SEQ ID NO: 103) and CDR3 (FQGSHVPPT , SEQ ID NO: 104) and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO: 105), CDR2 (WINTYTGEPTYADDFKG, SEQ ID NO: 106) and CDR3 (KGWMDFNSSLDY, SEQ ID NO: 107).

[0195] An hMN-15 antibody is a humanized antibody comprising the light chain variable region CDR sequences SASSRVSYIH (SEQ ID NO: 108); GTSTLAS (SEQ ID NO: 109); and QQWSYNPPT (SEQ ID NO: 110); and DYYMS heavy chain variable region CDR sequences (SEQ ID NO: 111); FIANKANGHTTDYSPSVk G (SEQ ID NO: 112); and DMGIRWNFDV (SEQ ID NO: 113).

[0196] In another example, therapeutic conjugates comprising an anti-CD74 antibody (eg, hLL1, milatuzumab, described in US Patent Nos. 7,074,403; 7,312,318; 7,772,373; 7,919,087 and 7,931,903) can be used to treat any of a variety of cancers that express CD74, including, but not limited to, kidney, lung, intestinal, stomach, breast, prostate, or ovarian cancer, as well as various hematological cancers, such as multiple myeloma, chronic lymphocytic leukemia, lymphoblastic leukemia acute, non-Hodgkin's lymphoma and Hodgkin's lymphoma. An hLL1 antibody is a humanized antibody comprising the light chain CDR sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO: 114), CDR2 (TVSNRFS; SEQ ID NO: 115) and CDR3 (SQSSHVPPT; SEQ ID NO: 116) and the sequences of heavy chain variable region CDRs CDR1 (NYGVN; SEQ ID NO: 117), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO: 118) and CDR3 (SRGKNEAWFAY; SEQ ID NO: 119).0197*

[0197] In another example, therapeutic conjugates comprising an anti-CD22 antibody (eg, hLL2, epratuzumab, described in US Patent Nos. 5,789,554; 6,183,744; 6,187,287; 6,306,393; 7,074,403 and 7,641,901 , or the chimeric or humanized antibody RFB4) can be used to treat any of a variety of cancers that express CD22 , including but not limited to indolent forms of B-cell lymphomas, aggressive forms of B-cell lymphomas B, chronic lymphatic leukemias, acute lymphatic leukemias, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma, follicular lymphoma, or diffuse B-cell lymphoma. Anti-CD22 conjugates are also used to treat autoimmune diseases, such as acute immune thrombocytopenia, thrombocytopenia chronic immune system, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, pemphigus bolosus, pemphigus vulgaris, diabetes mellitus (eg, juvenile diabetes), Henoch-Schonlein purpura, nephritis poststreptococcal, erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, syndrome Sjogren's, primary biliary cirrhosis, hashimoto's dermatitis/thyroxiditis active hepatitis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, graft-versus-host disease (GVHD) , organ transplant rejection, sepsis, septicemia, and inflammation. An hLL2 antibody is a humanized antibody comprising the light chain CDR sequences CDR1 (KSSQSVLYSANHKYLA, SEQ ID NO: 120), CDR2 (WASTRES, SEQ ID NO: 121) and CDR3 (HQYLSSWTF, SEQ ID NO: 122) and sequences of Heavy chain CDRs CDR1 (SYWLH, SEQ ID NO: 123), CDR2 (YINPRNDYTEYNQNFKD, SEQ ID NO: 124) and CDR3 (RDITTFY, SEQ ID NO: 125).

[0198] In one example, therapeutic conjugates comprising anti-CSAp antibodies, such as the hMu-9 MAb, can be used to treat colorectal, as well as pancreatic and ovarian cancers as described in US Patent Nos. 6,962,702; 7,387,772; 7,414,121; 7,553,953; 7,641,891 and 7,670,804. An hMu-9 antibody is a humanized antibody comprising the light chain CDR sequences CDR1 (RSSQSIVHSn Gn TYLE, SEQ ID NO: 126), CDR2 (KVSNRFS, SEQ ID NO: 127), and CDR3 (FQGSRVPYT, SEQ ID NO: 128). ) and variable chain CDR sequences CDR1 (EYVIT, SEQ ID NO: 129), CDR2 (EIYPGSGSTSYNEKFK, SEQ ID NO: 130), and CDR3 (EDL, SEQ ID NO: 131).

[0199] Therapeutic conjugates comprising the hPAM4 MAb can be used to treat pancreatic cancer or other solid tumors, as described in US Patent Nos. 7,238,786 and 7,282,567. An hPAM4 antibody is a humanized antibody comprising the CDR1 light chain variable region CDR sequences (SASSSVSSSYLY, SEQ ID NO: 132); CDR2 (STSNLAS, SEQ ID NO: 133); and CDR3 (HQWNRYPYT, SEQ ID NO: 134); and CDR1 heavy chain CDR sequences (SYVLH, SEQ ID NO: 135); CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO: 136) and CDR3 (GFGGSYGFAY, SEQ ID NO: 137).

[0200] In another example, therapeutic conjugates comprising an anti-AFP MAb antibody, such as IMMU31, can be used to treat hepatocellular carcinoma, germ cell tumors, and other AFP-producing tumors using humanized, chimeric, and antibody forms. humans, as described in US Patent No. 7,300,655. An IMMU31 antibody is a humanized antibody comprising the heavy chain CDR sequences CDR1 (SYVIH, SEQ ID NO: 138), CDR2 (YIHPYNGGTKYNEKFKG, SEQ ID NO: 139) and CDR3 (SGGGDPFAY, SEQ ID NO: 140) and the chain light CDR1 (KASQDINKYIG, SEQ ID NO: 141), CDR2 (YTSALLP, SEQ ID NO: 142) and CDR3 (LQYDDLWT, SEQ ID NO: 143).

[0201] In another example, therapeutic conjugates comprising an anti-HLA-DR MAb antibody, such as hL243, can be used to treat lymphoma, leukemia, cancers of the skin, esophagus, stomach, colon, rectum, pancreas, lung, breast, ovary, bladder, endometrium, cervix, testes, kidney, liver, melanoma, or other HLA-DR-producing tumors, as described in US Patent No. 7,612,180. An hL243 antibody is a humanized antibody comprising the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO: 144), CDR2 (WINTYTREPTYADDFKG, SEQ ID NO: 145) and CDR3 (DITAVVPTGFDY, SEQ ID NO: 146) and CDR of light chain sequences CDR1 (RASENIYSNLA, SEQ ID NO: 147), CDR2 (AASNLAD, SEQ ID NO: 148) and CDR3 (QHFWTTPWA, SEQ ID NO: 149).

[0202] In another example, therapeutic conjugates comprising an anti-CD20 MAb antibody, such as veltuzumab (hA20), 1F5, obinutuzumab (GA101), or rituximab, can be used to treat lymphoma, leukemia, immune thrombocytopenic purpura , systemic lupus erythematosus, Sjogren's syndrome, Evans syndrome, arthritis, arteritis, pemphigus vulgaris, kidney graft rejection, heart graft rejection, rheumatoid arthritis, Burkitt's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, small lymphocytic lymphoma, diffuse lymphoma B cell, marginal zone lymphoma, lymphocytic leukemia, acute lymphocytic leukemia, type I diabetes mellitus, GVHD, multiple sclerosis or multiple myeloma, as described in US patent numbers 7,435,803 or 8,287,864. An hA20 antibody (veltuzumab) is a humanized antibody comprising the light chain CDR sequences CDRL1 (Ra SSSVs Y iH, SEQ ID NO: 150), CDRL2 (ATSNLAS, SEQ ID NO: 151) and CDRL3 (QQWTSNPPT, SEQ ID NO : 152) and CDR chain sequences CDRH1 (SYNMH, SEQ ID NO: 153), CDRH2 (AIYPGNGDTSYNQKFKG, SEQ ID NO: 154) and CDRH3 (STYYGGDWYFDV, SEQ ID NO: 155).023*

[0203] In another example, therapeutic conjugates comprising an anti-CD 19 MAb antibody, such as hA19, can be used to treat B-cell-related lymphomas and leukemias, such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, or acute lymphoblastic leukemia. Other disease states that can be treated include autoimmune diseases, such as acute or chronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schon purpura , poststreptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodilia thrombosis syndrome, biliary bilitis surgery, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosing alveolitis, as described in US Patent Nos. 7,109,304, 7,462,352, 7,902,338, 8,147,831, and 8,337,840. An hA19 antibody is a humanized antibody comprising the light chain CDR sequences CDR1 KASQSVDYDGDSYLN (SEQ ID NO: 156); CDR2 DASNLVS (SEQ ID NO: 157); and CDR3 QQSTEDPWT (SEQ ID NO: 158) and heavy chain CDR sequences CDR1 SYWMN (SEQ ID NO: 159); CDR2 QIWPGDGDTNYNGKFKG (SEQ ID NO: 160) and CDR3 RETTTVGRYYYAMDY (SEQ ID NO: 161).

[0204] Therapeutic conjugates comprising anti-tenascin antibodies can be used to treat hematopoietic and solid tumors, and conjugates comprising the antibodies to tenascin can be used to treat solid tumors, preferably brain cancers such as glioblastomas.

[0205] Preferably, the antibodies that are used in the treatment of human disease are humanized (CDR-grafted) versions of antibodies; although murine and chimeric versions of antibodies can be used. IgG molecules of the same species as the delivery agents are primarily preferred to minimize immune responses. This is particularly important when considering repeat treatments. For humans, a humanized or human IgG antibody is less likely to generate an anti-IgG immune response in patients. Antibodies such as hLL1 and hLL2 are rapidly internalized after binding to the internalizing antigen on target cells, which means that the transported chemotherapeutic drug is also rapidly internalized into cells. However, antibodies that have slower internalization rates can also be used to effect selective therapy.

[0206] Therapeutic conjugates can be used against pathogens, as antibodies against pathogens are known. For example, antibodies and antibody fragments that specifically bind to markers produced by or associated with infectious lesions, including viral, bacterial, fungal and parasitic infections, for example caused by pathogens such as bacteria, rickettsiae, mycoplasmas, protozoa, fungi and viruses, and antigens and products associated with such microorganisms have been described, among others, in Hansen et al., US Patent No. 3,927,193 and US Patent 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709 and 4,624,846, and in Reichert and Dewitz, cited above. In a preferred embodiment, the pathogens are selected from the group consisting of HIV viruses, Mycobacterium tuberculosis, Streptococcus agalactiae, Methicillin- resistant Staphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans. , Histoplasma capsulatum, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II , human serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein virus -Barr, murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbis virus, d e lymphocytic choriomeningitis, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruz', Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosomabesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena , Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma artritidis, M. hyorhinis, M. oralema, M. salivarium, and M. pneumoniae, as described in US Patent No. 6,440,416.

[0207] Drug conjugates comprising anti-gp120 and other such anti-HIV antibodies can be used as therapeutic agents for HIV in AIDS patients; and Mycobacterium tuberculosis antibody drug conjugates are suitable as therapeutic agents for drug-refractory tuberculosis. Fusion proteins of anti-gp120 MAb (anti-HIV MAb) and a toxin, such as Pseudomonas exotoxin, have been examined for their antiviral properties (Van Oigen et al., J Drug Target, 5:75-91, 1998 ). Attempts to treat HIV infection in AIDS patients have failed, possibly due to insufficient efficacy or unacceptable host toxicity. Drug conjugates advantageously lack such toxic side effects of protein toxins and are therefore advantageously used in the treatment of HIV infection in AIDS patients. These drug conjugates can be administered alone or in combination with other antibiotics or therapeutic agents that are effective in such patients when administered alone. Candidate anti-HIV antibodies include the P4/D10 anti-envelope antibody described by Johansson et al. (AIDS. 2006 Oct 3; 20(15):1911-5), as well as anti-HIV antibodies disclosed and sold by Polymun (Vienna, Austria), also disclosed in US Patent 5,831,034, the US Patent 5,911,989 and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob. Chemother Agents. 2006; 50 (5): 1773-9. A preferred targeting agent for HIV is various combinations of these antibodies to overcome resistance.028*

[0208] More efficient incorporation into cells and pathogens can be achieved by the use of multivalent, multispecific, or multivalent, monospecific antibodies. Examples of such bivalent and bispecific antibodies are found in US Patent Nos. 7,387,772; 7,300,655; 7,238,785; and 7,282,567. These antibodies Multivalent or multispecific are particularly preferred in the targeting of cancers and infectious organisms (pathogens), which express multiple antigenic targets and even multiple epitopes of the same target antigen, but which often evade antibody targeting and sufficient binding for immunotherapy due to a insufficient expression or availability of a single antigen target on the cell or pathogen. By targeting multiple antigens or epitopes, such antibodies exhibit increased binding and residence time on the target, thus providing greater saturation with the targeted drug in this invention.

[0209] The therapeutic conjugates can also be used to treat autoimmune disease or dysfunction of the immune system (eg, graft-versus-host disease, organ transplant rejection). Antibodies for use in treating autoimmune/immune dysfunction disease may bind to exemplary antigens including, but not limited to BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43, CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD74, CD79a, CD79b, CD117, CD138, FMC-7, H2B, H3, H4, HLA-DR and MIF. Antibodies that bind to these and other target antigens, discussed above, can be used to treat autoimmune or immune dysfunction diseases. Autoimmune diseases that can be treated with immunoconjugates may include acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, meliglandular diabetes, purple hay Eskimo syndromes, poststreptococcal nephritis , erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, Sjogren's, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, bullous pemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, dorsal lateral scleroderosis, arteritis/polymyalgia d e giant cells, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis or fibrosing alveolitis.

[0210] The skilled artisan will appreciate that the subject immunoconjugates, comprising a 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, second antibody fragment, second immunoconjugate, radionuclide, toxin, drug, chemotherapeutic agent, radiotherapy, chemokine, cytokine, immunomodulator, enzyme, hormone, oligonucleotide, RNAi, or siRNA. Such additional therapeutic agents may be administered separately, in combination with, or bound to the subject antibody-drug immunoconjugates.

[0211] A therapeutic agent used in combination with the camptothecin conjugate of the present invention may comprise one or more isotopes. Radioactive isotopes useful for treating diseased tissue include, but are not limited to: 111In,

177Lu, 212Bi, 213Bi, 211At, 62Cu, 67Cu, 90Y, 125I, 131I, 32P, 33P, 47Sc, 111Ag, 67Ga, 142Pr, 153Sm, 161Tb, 166Dy, 189Re, 212Pb, 7AFe, 223Ra, 7A, 52,223Ac 89Sr, 99Mo, 105Rh, 109Pd, 143Pr, 149Pm, 169Er, 194Ir, 198Au, 199Au, 22 therapeutic radionuclide preferably has a decay energy in the range of 20 to 6,000 keV, preferably in the range of 60 to 200 keV for an emitter Auger, 100-2,500 keV for a beta emitter and 4,000-6,000 keV for an alpha emitter. The maximum decay energies of useful beta particle emitting nuclides are preferably from 20 to 5000 keV, more preferably from 100 to 4000 keV and most preferably from 500 to 2500 keV.

Radionuclides that substantially disintegrate with Auge-emitting particles are also preferred. For example,

Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-Ill, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. The decay energies of useful beta particle emitting nuclides are preferably <1,000 keV, more preferably

<100 keV and most preferably <70 keV. Radionuclides that decay substantially with the generation of alpha particles are also preferred. Such radionuclides include, among others: 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 useful alpha particle emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV. Other potential radioisotopes for use include 11C, 13N, 15O, 75Br, 198Au, 224Ac, 126I,

133I, 77Br, 113mIn, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 105Rh,

142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201Tl, 225Ac, 76Br, 169Yb and the like.

[0212] Radionuclides and other metals can be supplied, e.g. eg, using chelating groups attached to an antibody or conjugate. Macrocyclic chelates such as NOTA, DOTA, and TETA are used with a variety of metals and radiometals, most particularly radionuclides of gallium, yttrium, and copper, respectively. Such metalchelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates, such as macrocyclic polyethers, can be used to form complexes with 223Ra.

[0213] Therapeutic agents for use in combination with the camptothecin conjugates described herein also include, e.g. eg, chemotherapeutic drugs, such as vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotic, antiangiogenic, and proapoptotic agents, particularly doxorubicin, methotrexate, taxol, others camptothecins and others of these and other classes of anticancer agents and the like. Other cancer chemotherapeutic drugs include nitrogen mustards, alkylsulfonates, 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 in 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.

[0214] Exemplary drugs of use 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, crizotinibide, cyclophosphide agents estrogen receptor-binding receptor, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, inhibit protein transferase agents farnesil, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM- A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomicin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC), transplatin, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vincablastine, and vinylacridine. Such agents may be part of the conjugates described herein or alternatively may be administered in combination with the conjugates described, either before, simultaneously with or after the conjugate. Alternatively, one or more naked therapeutic antibodies as known in the art may be used in combination with the described conjugates. Exemplary naked therapeutic antibodies are described above.

[0215] Therapeutic agents that can be used in concert with camptothecin conjugates can also comprise toxins conjugated with targeting moieties. Toxins that can be used in this regard include ricin, abrin, ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, phytolac antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. (See, e.g., 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 those are known to those skilled in the art and are described in US 6,077,499.

[0216] Another class of therapeutic agent may comprise one or more immunomodulators. The immunomodulators for use may be selected from a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony-stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin, and a combination thereof. Specifically 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 hematopoietic factor. colonies of granulocytes and macrophages (GMCSF), interferon, such as interferons-a, -p, -i -A, and stem cell growth factor, such as the so-called "S1 factor". 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); liver growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-a and -p; Muller-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-p; platelet growth factor; transforming growth factors (TGFs) such as TGF-a and TGF-p; insulin-like growth factor I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -p, -y and -A; colony stimulating factors (CSF) such as macrophage-CSF (M-CSF); interleukins (IL) such as IL-1, IL-1a, 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 recombinant cell culture and biologically active equivalents of native sequence cytokines.

[0217] Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10.

Formulation and administration

[0218] Suitable routes of administration of the conjugates include, without limitation, oral, parenteral, rectal, transmucosal, intestinal administration, intramedullary, intrathecal intraventricular, direct, intravenous, or intraperitoneal injections. The preferred routes of administration are parenteral. Alternatively, the compound may be administered locally rather than systemically, e.g. eg, by injecting the compound directly into a solid tumor.

[0219] Immunoconjugates can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the immunoconjugate is combined in admixture with a pharmaceutically acceptable excipient. suitable. Sterile phosphate buffered saline is an example of a suitable pharmaceutical carrier. Other suitable excipients are well known to those skilled in the art. See, p. eg, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th edition (Lea and Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition (Mack Publishing Company 1990) and revised editions thereof.

[0220] Preferably, the immunoconjugate is formulated in Good'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 [Tubes]. The most preferred buffers are MES or MOPS, preferably in the concentration range of 20 to 100 mM, more preferably around 25 mM. Most preferred is 25 mM MES, pH 6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/v polysorbate 80 as excipients, with the final buffer concentration modified to 22.25 mM as a result of the addition of excipients. The preferred method of storage is as a lyophilized formulation of the conjugates, stored in the temperature range of -20°C to 2°C, with 2°C to 8°C being most preferred storage.

[0221] The immunoconjugate may be formulated for intravenous administration via, e.g. g., bolus injection, slow infusion, or continuous infusion. Preferably, the antibody of the present invention is infused 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 in 30 minutes, preferably even 15 minutes, and the rest can be infused over the next 2-3 hours. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. g., sterile pyrogen-free water, before use.

[0222] Additional pharmaceutical methods may be employed to control the duration of action of the therapeutic conjugate. Controlled release preparations can be prepared by using polymers to complex or adsorb the immunoconjugate. For example, biocompatible polymers include poly(ethylene-covinyl) acetate matrices and matrices of a polyanhydride copolymer of a dimer of stearic acid and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release of an immunoconjugate from such a matrix depends on the molecular weight of the immunoconjugate, the amount of immunoconjugate within the matrix, and the size of the dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., Supra. Other solid dosage forms are described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th edition (Lea and Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition (Mack Publishing Company 1990), and revised editions thereof.

[0223] Generally, the dosage of an immunoconjugate administered to humans will vary depending on factors such as the patient's age, weight, height, gender, general medical condition, and prior medical history. It may be desirable to provide the recipient with a dose of immunoconjugate that is in the range of about 1 mg/kg to 24 mg/kg as a single intravenous infusion, although a lower or higher dose may also be administered as circumstances require. A dose of 1 to 20 mg/kg for a 70 kg patient, e.g. eg, it is 70 to 1,400 mg or 41 to 824 mg/m2 for a 1.7-m patient. The dose can be repeated as needed, e.g. For example, once a week for 4 to 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, as needed for maintenance therapy. Preferred doses may include, but are not limited to, 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 of 1 to 24 mg/kg can be used. The dose is preferably administered several times, once or twice a week. A minimum dosage schedule of 4 weeks, more preferably 8 weeks, most preferably 16 weeks or more may be used. The administration schedule may comprise administration once or twice a week, on 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.

[0224] Alternatively, an immunoconjugate may be administered as a dose every 2 or 3 weeks, repeated for a total of at least 3 dosages. Or twice a week for 4-6 weeks. If the dose is reduced to approximately 200-300 mg/m2 (340 mg per dose for a 1.7 m patient, or 4.9 mg/kg for a 70 kg patient), it can be administered once or even twice. per week for 4 to 10 weeks. Alternatively, the dosage schedule can be reduced, ie every 2-3 weeks for 2-3 months. However, it has been found that even higher doses, such as 12 mg/kg once a week or once every 2-3 weeks, can be given by slow intravenous infusion, for repeated dosing cycles. The dosage schedule may optionally be repeated at other intervals and the dosage may be administered via various parenteral routes, with appropriate adjustment of dosage and schedule.

[0225] Preferably, the immunoconjugates are of use for cancer therapy. Examples of cancers include, but are not limited to carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More particular examples of such cancers are listed below and include: squamous cell cancer (eg, epithelial squamous cell cancer), Ewing's sarcoma, Wilms tumor, astrocytomas, lung cancer including squamous cell lung cancer. non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, cancer of the stomach or gastric including gastrointestinal cancer, cancer of the pancreas, glioblastoma multiforme, cancer of the cervix, ovarian cancer, cancer liver, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors, medullary thyroid cancer, differentiated thyroid carcinoma, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial cancer or carcinoma of the uterus, carcinoma of salivary glands, kidney or kidney cancer, prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, as well as head and neck cancer. The term "cancer" includes primary malignant tumors or cells (e.g., those whose cells have not migrated to sites in the subject's body other than the original tumor or malignancy site) and secondary malignant tumors or cells (e.g., eg, those arising from metastases, the migration of malignant cells, or tumor cells to secondary sites that are different from the original tumor site).

[0226] Other examples of cancers or malignancies include, but are not limited to: childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, hepatocellular cancer in adults (primary), liver cancer in adult (primary), adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin lymphoma, adult lymphocytic leukemia, adult non-Hodgkin lymphoma, primary adult liver cancer, adult soft tissue sarcoma, related lymphoma with AIDS, AIDS-related malignancies, anal cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumors, breast cancer, renal pelvis and ureter cancer, lymphoma of the central nervous system (primary), central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, liver cancer childhood leukemia (primary), childhood liver cancer (primary), childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brainstem glioma, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, childhood extracranial germ cell tumors, childhood Hodgkin disease, Childhood Hodgkin lymphoma, childhood hypothalamic and lymphatic visual pathway glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin lymphoma, childhood supratentorial and pineal primitive neuroectodermal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cell cancer, cutaneous colon lymphoma, pancreatic islet cell endocrine carcinoma, endometrial cancer, ependymoma, epithelial cancer, esophageal cancer , Ewing's sarcoma and tumors r related, exocrine pancreatic cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher disease, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, bowel cancer, intraocular melanoma, cell carcinoma of the islets, islet cell pancreatic cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma , malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic occult primary squamous neck cancer asiatic, metastatic primary squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma/plasma cell neoplasia, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, nasal cavity and sinus and paranasal cancer, nasopharyngeal cancer, neuroblastoma , non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, occult primary metastatic squamous neck cancer, oropharyngeal cancer, malignant fibrous sarcoma of bone, osteosarcoma/malignant fibrous histiocytoma, osteosarcoma/malignant fibrous histiocytoma of bone , ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemias, polycythemia vera, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, primary central nervous system lymphoma, cancer primary liver cancer, prostate cancer, rectal cancer, kidney cancer, cancer renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoidosis sarcomas, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, supratentorial primitive neuroectodermal and pineal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis cancer and of the ureter, trophoblastic tumors, cell cancer of the ureter and renal pelvis, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms tumor and any other hyperproliferative disease, in addition to neoplasia, located in an organ system mentioned above.

[0227] The disclosed methods and compositions can be used to treat malignant or premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to the disorders described above. Said uses are indicated in known or suspected conditions of previous progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or, more particularly, dysplasia has occurred (for a review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2nd edition, WB Saunders Co., Philadelphia, pp. 68-79 (1976)).

[0228] Dysplasia is often a precursor to cancer and is found primarily in epithelia. It is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplasia characteristically occurs when there is chronic irritation or inflammation. Dysplastic disorders that may be treated include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital cranial dysplasia, craniocarpotarsal dysplasia, craniometaphyseal dysplasia, dentin dysplasia, diaphyseal dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, epiphysial dysplasia hemimelia, epiphysial dysplasia multiplex, strut dysplasia digita, epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of the jaw, fibromuscular dysplasia, bone fibrous dysplasia , florid bone dysplasia, hereditary renal retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphyseal dysplasia, Mondini dysplasia, di Monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphyseal dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, ophthalmomandibulomelic dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia, pseudoachondroplastic spondyloepiphyseal dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepidyl dysplasia ventriculo-radial.

[0229] Additional pre-neoplastic disorders that may 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, keratosis, Bowen's disease, farmer's skin, solar cheilitis and solar keratosis.

[0230] Preferably, the method of the present disclosure is used to inhibit the growth, progression and/or metastasis of cancers, in particular those listed above.

[0231] Additional hyperproliferative diseases, disorders and/or conditions include, but are not limited to progression, and/or metastases of malignant tumors and related disorders such as leukemia (including acute leukemias; eg, acute lymphocytic leukemia, acute myeloid leukemia [including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia]) and chronic leukemias (eg, chronic myelocytic [granulocytic] leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (eg, Hodgkin's disease and multiple myeloid) Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelium, leiomyosarcoma, rhabdomyosarcoma, carcinoma of the colon, pancreatic cancer, breast cancer, or varicose cancer, cancer prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, duct bicarcinoma, seminoma carcinoma hepatorium, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, meningroglioma acoustic, neuroblastoma and retinoblastoma.

[0232] Autoimmune diseases that can be treated with immunoconjugates may include acute and immune chronic thrombocytopenias, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, purpura Henoch-Schonlein, poststreptococcal nephritis, erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing thrombosis syndrome syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, bullous pemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, emyotrophic lateral sclerosis, tabes dorsalis, large cell arteritis/polymyalgia , perennial anemia iciosa, rapidly progressive glomerulonephritis, psoriasis or fibrosing alveolitis.

kits

[0233] The instant disclosure relates to kits containing components suitable for treating diseased tissue in a patient. Exemplary kits may contain at least one conjugated antibody or other targeting moiety as described herein. If the composition containing the components for administration is not formulated for administration via the gastrointestinal tract, such as oral administration, a device capable of administering the kit components via some other route may be included. One type of device, for applications such as parenteral administration, is a syringe used to inject the composition into the body of a subject. Inhalation devices can also be used.

[0234] The kit components may be packaged together or separately in two or more containers. In some In embodiments, the containers may be vials containing sterile lyophilized formulations of a composition that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents. Other containers that can be used include, but are not limited to a bag, tray, box, tube, or the like. Kit components can be packaged and kept sterile within containers. Another component that may be included is instructions to a person using a kit for its use.

EXAMPLES

general

[0235] Abbreviations used below are: DCC, dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; EEDQ, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; MMT, monomethoxytrityl; PABOH, p- aminobenzyl alcohol; PEG, polyethylene glycol; SMCC, succinimidyl 4-(W-maleimidomethyl)cyclohexane-1-carboxylate; TBAF, tetrabutylammonium fluoride; t Bd MS, tert-butyldimethylsilyl chloride.

[0236] The hydroxy compound chloroformates in the following examples were prepared using triphosgene and DMAP according to the procedure described in Moon et al. (J. Medicinal Chem. 51: 6916-6926, 2008). Extractive work-up refers to extraction with chloroform, dichloromethane, or ethyl acetate and optionally washing with saturated bicarbonate, water, and saturated sodium chloride. Flash chromatography was performed using 230-400 mesh silica gel and methanedichloromethane gradient, using up to 15% v/v methanol-dichloromethane, unless otherwise noted. Reversed-phase HPLC was carried out by Method A using a 7.8 x 300 mm C18 HPLC column, equipped with a precolumn filter, and using a solvent gradient from solvent A to 100% solvent B in 10 minutes to 100%. at a flow rate of 3 ml per minute and maintaining 100% solvent B at a flow rate of 4.5 ml per minute for 5 or 10 minutes; or by Method B using a 4.6 x 30 mm, 2.5 pm Xbridge C18 column equipped with a pre-column filter, using the solvent gradient from 100% solvent A to 100% solvent B at a flow rate 1.5 ml per minute for 4 min and 100% solvent B at a flow rate of 2 ml per minute for 1 minute. Solvent A was 0.3% aqueous ammonium acetate, pH 4.46, while solvent B was 9:1 acetonitrile-aqueous ammonium acetate (0.3%), pH 4.46. HPLC was monitored by an on-line dual absorbance detector set at 360nm and 254nm.

Example 1: Preparation of CL2A-SN-3

[0237] A preferred reaction scheme for the synthesis of CL2A-SN-38 is shown in FIG. 1. An earlier version of the synthetic protocol, described in US Patent No. 9,107,960, was the basis for the improved protocol shown in FIG. 1. The substrates, intermediates, and reactions described in US Patent No. 9,107,960 are discussed in this Example. Additional improvements to the protocol of US Patent No. 9,107,960, incorporated in FIG. 1, are described in the following examples.

[0238] Preparation of O-(2-Azidoethyl)-O'-[(N-diglycolyl-2-aminoethyl)-Lys(MMT)-PABOHlheptaethyleneglycol (intermediate 3): In a 500 mL single neck flask, commercially available Fmoc-Lys(MMT)-OH (16 g), p-aminobenzyl alcohol (3.26 g) and EEDQ (6.52 g) were added, followed by anhydrous dichloromethane (80 mL). After stirring overnight, diethylamine (25 mL) was added and after an additional 6 h the reaction mixture was concentrated to ~50 mL volume. This was diluted with heptane and the solution concentrated back to 50 mL. Two additional trials with heptane (50 ml each) gave a biphasic mixture containing gummy material at the bottom. The gummy material was taken up in dichloromethane (24 ml), stirred and treated with slow addition of heptane (80 ml). After stirring for 1 h, the suspension was filtered to obtain 13.02 g (99.6% yield) of Lys(MMT)-PABOH (intermediate 2, Figure 1). The material was optionally further purified by chromatography if residual diethylamine was present. Lys(MMT)-PABOH (11.51g) was mixed with a solution of O-(2-azidoethyl)-O'-(N-diglycolyl-2-aminoethyl)heptaethyleneglycol (PEG-N3; 12.107g) in anhydrous dichloromethane ( 90ml). To this stirred solution EEDQ (5.54 g) was added. After ca. 18 h, the reaction mixture was concentrated and purified by silica gel chromatography, using ethyl acetate-methanol gradient elution, to obtain the pure title product (intermediate 3, Figure 1).

[0239] Preparation of TBDMS-SN-38 (intermediate 4): The preparation was modified with the use of dichloromethane, instead of dimethylformamide, as solvent, which allowed easier work up. A solution of diisopropylethylamine (36.6 g) in anhydrous dichloromethane (160 mL) was added to SN-38 (33 g). The suspension was cooled in an ice/water bath and stirred. To this stirred suspension was added a solution of tert-butyldimethylsilyl chloride (31.72 g) in dichloromethane (125 ml). The cold bath was removed and the reaction mixture was stirred for 4 h. The clear reaction mixture was washed with 380 mL of 0.2M HCl in 10% sodium chloride solution and 300 mL of 10% sodium chloride solution. The product, after drying and removal of solvent, was precipitated from ethyl acetate-heptane to obtain 38.75 g (91% yield) of TBDMS-Sn-38.

[0240] Generation of 10-0-TBDMS-SN-38-20-0-chloroformate (reactive intermediate 5) and the preparation of O-(2-azidoethyl)-O'-r(N-diglycolyl-2-aminoethyl) -Lys(MMT)-PABOCO-20-0-SN-38lheptaethylene glycol (intermediate 7): To avoid a large exotherm from the bolus addition of the required amounts of triphosgene, an improved process was invented by addition of a solution of triphosgene (0.24 g) in anhydrous dichloromethane (4 mL), over 30 minutes, to a stirred mixture of 0.94 g of TBDMS-SN-38 and DMAP (0.76 g) in dichloromethane ( 17ml). This resulted in a 98.5% conversion to a reactive intermediate (5 in Figure 1), as determined by HPLC analysis of a quenched aliquot of the reaction mixture, quenched with anhydrous methanol. This ensured clean formation of the required product, with no evidence of the formation of a dimeric material due to rapid cooling of the initially formed chloroformate with unreacted SN-38. Although the bolus addition resulted in a large exotherm from 5.8°C to 17.6°C, the slow addition maintained the internal temperature at <10°C throughout, without compromising reaction quality. The same approach was repeated using 11.25 g (22.2 mmol) of TBDMS-SN-38 in 250 ml of anhydrous dichloromethane and using other reagents proportionally as described for the small scale reaction above. After 15 min, a solution of 25.01 g of O-(2-Azidoethyl)-O'-[(N-diglycolyl-2-aminoethyl)-Lys(MMT)-PABOH]heptaethylene glycol (intermediate 3, Figure 1) in dichloromethane (125 mL) was added over 8 min. After 75 min, the reaction mixture was washed sequentially with 50 mM sodium acetate buffer, pH5.3, water, and brine, and dried over anhydrous sodium sulfate. The product solution was then shaken with a mixture of 1M tetrabutylammonium fluoride in tetrahydrofuran (31 ml), anhydrous dichloromethane (34 ml) and acetic acid (3 ml). After 2h, the reaction mixture was washed with 0.25M citrate buffer, pH 6, water, and brine, and dried over anhydrous sodium sulfate. Silica gel chromatography, gradient elution with methanol-methylene chloride mixtures afforded 17.3 g (52% yield) of the title product (intermediate 7).

[0241] Preparation of CL2A-SN-38: O-(2-azidoethyl)-O'-[(N-diglycolyl-2-aminoethyl)-Lys(MMT)-PABOCO-20-0-SN-38]heptaethyleneglycol ( intermediate 7, 10.2 g) in anhydrous dichloromethane (130 mL) was mixed with 4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (intermediate 8 ['MCC-yne']; 3 .78g). To this was added a mixture of triphenylphosphine (0.38g), cuprous bromide (0.24g) and diisopropylethylamine (0.25ml) in dichloromethane (100pl). The reaction mixture was stirred at room temperature for 14h. The material was concentrated and purified by chromatography; the pure product solution was washed with EDTA, water and brine. The product, O-{2-[(1,2,3-triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-carboxamidomethyl]]ethyl}-O'-[(N-diglycolyl-2- aminoethyl)-Lys(MMT)-PABOcO-20-0-SN-38]heptaethylene glycol (intermediate 9) was obtained in the amount of 10.8 g (89% yield), which was better than the maximum yield of 67%. previously obtained. This modified procedure improved yield due to reduced reaction time (14 h vs. 18-41 h) and also avoided a stability problem previously encountered with intermediate 9 by performing chromatography first and EDTA extraction later. The product solution in dichloromethane was mixed with anisole (2.8 g), cooled to <5°C and reacted with dichloroacetic acid (5.8 g) for 2 h. The final product, CL2A-SN-38, was precipitated from tert-butyl methyl ether in 81% total yield (2 steps).

Example 2. Improved Protocol for Synthesis of CL2A-SN-38

[0242] While the synthesis protocol described in Example 1 above is suitable for the large-scale preparation of CL2A-SN-38, further improvements are disclosed herein that were not known and might not have been predicted. the filing date of 9,107,960.

[0243] The CL2A linker described herein comprises a PEG moiety containing a defined number of PEG monomers. As shown in FIG. 1, in a preferred embodiment, the number of PEG monomers used in the linker is eight. However, alternative embodiments within the scope of the invention may use a number of PEG monomers between 1-30, more preferably 1-12, more preferably 6-10, more preferably 8.

[0244] There are 3 primary stages in which the process improvements were made: (a) in the conversion of Intermediate 6 to Intermediate 7; (b) in the conversion of intermediate 8 to intermediate 9; and (c) in the conversion of intermediate 9 to intermediate 10.

Yield improvement in the preparation of intermediate product 7

[0245] In the conversion of silyl-protected intermediate 6 to desilylated intermediate 7, the tetrabutylammonium fluoride used was changed from 1.4-fold molar excess in previous versions to 1.1-fold molar excess in the present protocol. This level of reagent was equally effective and also reduced the impurity profile, thus improving the purity profile of Intermediate 7 and leading to improved yield.

[0246] The aqueous treatments in the preparations of both Intermediates 6 and 7 were reduced as follows. In previous versions, the treatment consisted of washing the organic extracts four times with 0.05 M sodium acetate buffer, pH5.3 for intermediate 6 and four times with 0.25 M citrate buffer, pH 6 for intermediate 7, with washed with water subsequently in each case. In the improved protocol, the buffer washes were reduced to 3 and 2 for intermediates 6 and 7, respectively, and the water washes were eliminated in both cases. These modifications considerably reduced emulsion formation and improved product recovery in the organic extract.

[0247] Chromatographic purification of intermediate 7 previously involved the use of dichloromethane-methanol mixtures for elution. In the improved protocol of FIG. 1, the elution was modified to a combination of dichloromethane-ethyl acetate-methanol mixtures with varying concentration of methanol, followed by varying concentration of dichloromethane-methanol mixtures. This resulted in better and faster separation of the intermission and improved recovery. The proportions of solvents used are discussed in more detail in Example 4 below.

[0248] The overall yield in the 3-step sequence from Intermediate 4 to Intermediate 7 was improved from the range of 29-40% to 64% by incorporating these process changes.

Improvement of the robustness of the process in the preparation of intermediate compound 9

[0249] In the protocol(s) described previously, the copper (+1) catalyzed reaction between intermediates 7 and 8 to produce intermediate 9 was performed using a newly formed complex of cuprous bromide and triphenylphosphine in dichloromethane with the addition of a tertiary amine, such as diisopropylethylamine. This procedure worked well, producing intermediate 9 in 50-60% yields after purification. However, the proportion of cuprous bromide and triphenylphosphine in the complex had to be carefully controlled. It was shown that in the presence of triphenylphosphine alone, the product quality of Intermediate 9 deteriorated over time, indicating the deleterious effect of this reagent, which required rapid removal of this reagent once the copper-catalyzed reaction was complete. . This introduces some limitation, with the potential to cause a reduction in the yield of purified intermediate 9, particularly in large scale preparations.

[0250] In the modified procedure described here, the reaction was performed using a biphasic mixture containing copper sulfate and ascorbic acid in water and intermediates 7 and 8 , as well as 2,6-collidine, in dichloromethane. On stirring the biphasic solution overnight, typically 18 h, the conversion was complete, and the crude product is separated from copper sulfate and ascorbate by a simple aqueous EDTA wash and purified by chromatography. Product yield was similar to cuprous bromide/triphenylphosphine as catalyst, but the improved protocol avoids the use of triphenylphosphine and its possible effects on intermediate stability.

Improvement of processes in the preparation of the final product 10 ( CL2A-SN-38)

[0251] The stability profile of intermediate 9 in solution and as an isolated material without solvent showed that the product maintains >97% purity, either stored as a solid product at -20°C for 4 days or as a solution in dichloromethane at room temperature for 1 day. In the latter case, storage at room temperature for 4 days reduced the purity to 89.4%, while the solution stored at -20°C showed only 92% purity. In the protocol described above, the material in dichloromethane, obtained after chromatography and washing with EDTA, was used as is for the deprotection reaction mediated by dichloroacetic acid (DCA) and anisole. In that situation, depending on the storage duration of intermediate 9 in solution form, the quality of the final material (CL2A-SN-38) varied. In the improved protocol, intermediate 9, after purification, was concentrated to a solid which was subsequently used in the final deprotection step. This modification removed uncertainty about changes in product quality of Intermediate 9 before the final deprotection step.

[0252] Another improvement related to the addition of the crude product solution, after the reaction, dropwise over several hours, in vigorously stirred tert-butyl methyl ether (t-BME), which allowed the precipitation of the product as a filterable solid. If the addition of the reaction mixture is not gradual and if the stirring is not vigorous, the product will be oiled instead of precipitating as a solid.

[0253] Other changes can be incorporated at various steps in the synthesis protocol of FIG. 1. For example, in the conversion of Intermediate 1 to Intermediate 2 and in the conversion of Intermediate 2 to Intermediate 3, EEDQ is shown as the amide coupling reagent. However, one of ordinary skill in the art will realize that amide coupling can be achieved using numerous alternative reagents, as reviewed in: Han SY and Kim YA. Recent development of peptide coupling reagents in organic synthesis. Tetrahedron 2004; 60: 2447-2467; and Valleur E and Bradley M. Amide bond formation: beyond the myth of coupling reagents. Chem Soc Rev 2009; 38: 606-631. In non-limiting examples, useful amide coupling reagents can be selected from: (1) carbodiimide reagents such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC ) used in conjunction with additives selected from 1-hydroxy-1H-benzotriazole (HOBt), N-hydroxysuccinimide (HOSu) and dimethylaminopyridine (DMAP); (2) 0-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and O-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium salts hexafluorophosphate (HBTU); (3) N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and N-isobutoxycarbonyl-2-isobutoxy-1,2-dihydroquinoline (IIDQ); and (4) isobutyl chloroformate.

Example 3. Use of 1.1 vs. 1.4 Equivalents of Tetrabutylammonium Fluoride (TBAF) for the Desilylation of Intermediate 6 to Intermediate 7

[0254] Crude intermediate 6 (13.78 g, 0.873 mmol) dissolved in 14 mL DCM was cooled and treated with 1.4 equivalents of 1 M tetrabutylammonium fluoride (TBAF) containing acetic acid (0.126 g). , with the reaction mixture maintained at < 20°C. After 1 h, the reaction mixture was washed with 0.25 M citrate buffer, pH 6 (3 x 10 mL), water, and 5% sodium chloride solution, and dried over anhydrous sodium sulfate. HPLC showed 85.5% purity for crude product 7, with impurities at four peak positions as follows: 1.0% (relative retention time or RRT of 0.79), 2.4% (RRT of 0.864), 1.5% (RRT of 0.94) and 3.7% (RRT of 1.162) When the same reaction was carried out using 1.1 equiv. From 1 M TBAF, the purity of the product by HPLC improved slightly to 88.2%, but the impurities decreased significantly: 0.3% (RRT of 0.79), 0.7% (RRT of 0.864), 0.8% (RRT of 0.94), and 2.4% ( RRR of 1.162). Improving these impurity profiles simplified subsequent chromatographic separation.

Example 4. Use of a ternary solvent system versus a binary solvent system in the chromatography of intermediate 7

[0255] A solution of 17.2 g of crude intermediate 7 was applied to a column of packed silica gel in mobile phase A (MPA), which was 15% dichloromethane (DCM)/ethyl acetate. Elution was by step gradient using 0-5% methanol-MPA, followed by a 2:1 mixture of 5% methanol-MPA and 5% methanol-DCM; a 1:2 mixture of 5% methanol/MPA and 5% methanol-DCM; and finally 5% methanol/DCM. Seven fractions collected showed a product purity of >93%, resulting in a 69.6% yield for the product versus a 44.7% yield for the binary chromatographic elution system described above. In the latter system, a 17.2 g solution of crude intermediate 7 was applied to a silica gel column under the same conditions as for the modified procedure, but with step gradient elution using 0-methanol-DCM mixtures. 5%. A product with >93% purity was obtained in only 2 fractions, resulting in a 44.7% yield. In subsequent experiments, MPA was prepared with 30% DCM/ethyl acetate, which was then mixed with varying concentrations of methanol for elution.

Example 5. Steps to improve the yield in the 3-step conversion of intermediate 4 to intermediate 7

[0256] A solution of 8.868 g (17.5 mmol) of TBDMS-SN-38 (intermediate 3) and 4-[N,N-dimethylamino]pyridine (DMAP; 7.06 g; 3.3 equiv.) in 133 ml of dichloromethane (DCM) was stirred under nitrogen and cooled to an internal temperature of 2.6°C. To this solution a solution of triphosgene (2.4 g, 8.088 mmol) in 44 ml of DCM was added over 6 minutes. The reaction mixture was heated to an internal temperature of 15°C for 20 min and kept at 15-16°C for 10 min. The reaction mixture was cooled to an internal temperature of 6.4°C, followed by the addition of a solution of intermediate 3 (18.03 mmol, 1.03 equiv.) in 97.5 mL of anhydrous DCM, while maintained the internal temperature at < 15°C. The reaction mixture was stirred for 3.5 h, washed with 50 mM sodium acetate, pH5.3, buffer (3 x 97.5 g) and 10% sodium chloride (97.5 g), and dried. over anhydrous sodium sulfate overnight.

[0257] The filtered solution was cooled to an internal temperature of < 15°C, and treated with a solution of 1M TBAF (19.3 mL, 19.3 mmol, 1.1 equiv) in 27 mL DCM containing acetic acid (2.52 g, 42.0 mmol, 2.4 equiv.). The mixture was stirred at 10-16°C for 45 min, and then washed with 2 x 300 g of a mixture of 400 g 0.25 M citrate buffer, pH 6, and 200 g 10% sodium chloride. % and dried over anhydrous sodium sulfate. The dried solution was concentrated to ~50 mL and applied to a 400 g column of silica gel packed in 30% DCM/ethyl acetate (MPA). Elution was carried out with a step gradient containing increasing concentration of methanol (1-5% methanol in MPA), followed by elution with 5% methanol in MPA/DCM 2:1, 5% methanol in MPA /DCM 1:2, and 5% methanol/DCM. The purest fractions, >94% pure, were combined, concentrated to ~50 mL, chased with ethyl acetate (EtOAc; 2 x 80 mL) and EtOAC/heptane (1:1; 160 mL) to volume. final 35-50 mL and diluted with 160 mL heptane. The precipitated solid product was filtered off and dried at 40°C under vacuum. The yield was 17.21 g (64.3% after adjustment for 3.2 wt% residual heptane). The HPLC purity of the product, intermediate 7, was 95%. 1H NMR spectrum confirmed the structure. The isolated yield of 64.3% was higher than the 29-40% obtained with the protocols described above.

Example 6. Stability of intermediate 9 in the presence of triphenylphosphine

[0258] Pure pooled chromatographic fractions of intermediate 9 were evaluated in the presence of 0 mol% (control), 10 mol%, and 20 mol% triphenylphosphine at room temperature over a period of 96 h. Table 6 below shows that the quality of the product deteriorated in the presence of triphenylphosphine.

Table 6. % HPLC purity of intermediate 9 (solution form) in the presence of triphenylphosphine (Ph3P) at room temperature

Example 7. Improvements for the preparation of intermediate 9

[0259] An amber bottle was charged with intermediate 7 (n=8; 232 g; 157 mmol; 1 equiv) and methylene chloride (2.1 kg) to form a solution. The solution was transferred to a 12 L round bottom flask equipped with a stirrer. top, a temperature probe and a nitrogen blanket. Intermediate 8 (77 g, 280 mmol, 1.8 equiv.), methylene chloride (1.2 kg), 1 M copper(II) sulfate solution (32 g), 1 M sodium L-ascorbate (+) solution (93 g) and 2,6-lutidine (20 g). The resulting solution was stirred overnight. After 18.5 h, the reaction mixture was washed six times with 1.5 L of 0.1 M EDTA solution, pH5.5. The organic layer was concentrated to 0.7 L and purified by flash chromatography on 6 kg of silica gel packed in an 11" wide column. Elution was carried out with mixtures of 0-8% methanol and sodium chloride. methylene in volumes of 5.8, 14.5, 14.5, 23.2, 23.2, 23.2, 23.2 and 46.4 liters for successive stepwise gradients The pure fractions were combined and the fractions evaporated. solvents Recovery: 145 g (52.6% yield; HPLC purity: 96.5%).

Example 8. Stability profile of intermediate 9

[0260] Intermediate 9 was prepared using a biphasic mixture of aqueous copper sulfate/sodium ascorbate and DCM solution of intermediates 7 and 8. Isolation of the product gave a 60-65% yield of pure intermediate 9. Both Solvent-free product (solid product) as well as the combined pure fractions from chromatography (solution form) were subjected to stability analysis. Table 7 provides stability data by HPLC analysis. From the data, it was determined that Intermediate 9 is best stored in solid form at low temperature prior to CL2A-SN-38 deprotection.

Table 7. % HPLC purity of intermediate 9 stored under various conditions

Example 9. Preparation of CL2A-SN-38 (final product 10)

[0261] Intermediate 9 (145 g) was dissolved in methylene chloride (1.7 kg), and the solution transferred to a 5-L round-bottom flask equipped with an overhead stirrer, a temperature probe, and a heating pad. nitrogen. Anisole (39 g, 359 mmol, 4.35 equiv) was added to the solution and the solution was cooled to 4°C. Dichloroacetic acid (107 g, 831 mmol, 10.08 equiv) was added over 20 min, keeping the temperature <4°C. After the addition, the temperature was increased to 15°C and the reaction mixture was stirred for 4h. This reaction mixture was added to tert-butyl methyl ether (t-BME; 3.8 kg), taken up in a 22 L round bottom flask equipped with overhead stirrer, addition funnel and nitrogen inlet, for 1. 5 h, maintaining vigorous stirring. The resulting suspension was stirred at room temperature for 12 h. The solids were allowed to settle and the supernatant was decanted. The solids were rinsed with 4% methylene chloride/tBME, slurried with t-BME, and filtered. The collected solids were dried at <40°C in vacuo to obtain 124.3 g of the title compound as a yellow solid (93.6% yield).

Example 10. Conjugation of CL2A-SN-38 to antibodies

[0262] The anti-CEACAM5 humanized MAb, hMN-14, the anti-CD22 humanized MAb, hLL2, the anti-CD20 humanized MAb, hA20, the anti-EGP-1 Humanized MAb, hRS7 were used in these studies and humanized anti-mucin MAb, hPAM4. Each antibody was slightly reduced with Tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffer at pH in the range 7-7.4, pH adjusted to 6.5, and reacted with ~10-fold molar excess. of CL2A-SN-38 using 5-10% v/v DMSO as cosolvent and incubating for 20 min at room temperature. Any excess thiol was capped with N-ethylmaleimide used as an aqueous solution in 10-fold molar excess relative to the antibody.

[0263] The conjugate was purified by tangential flow filtration (TFF), using 20-30 diafiltration volumes of the final formulation buffer, 25 mM MES, pH 6.5. This method avoided cumbersome sequential purification on hydrophobic and size exclusion columns, allowing hundreds of grams of conjugates to be easily purified. The product was analyzed for SN-38 by absorbance at 366nm and correlated with standard values. Protein concentration was deduced from the absorbance at 280 nm, corrected for the absorbance spillover of SN-38 at this wavelength. From these, the SN-38/MAb substitution ratios (DAR) were determined. Purified conjugates were stored as lyophilized formulations in glass vials, vacuum stoppered and stored in a -20°C freezer. The DARs obtained for some of these conjugates, which typically ranged from 5 to 7, are shown in Table 8.

T a b le 8: R e la tio n s o f S N -38 /drug M A b /M A b (D A R ) in some conju g a ts

Mp xis at 11. E fic ac ra ia s I p p u tics in vivo in the mode re c sp lín ic osdeca rc in omadep to LAGs co aso as an nh um or.

[0264] Immunocompromised athymic nude mice, bearing subcutaneous human pancreas or colon. Tumor xenografts were treated with a specific CL2A-SN-38 conjugate or control conjugate or left untreated. Therapeutic efficacies of the specific conjugates were observed. FIG. 2 shows a Capan 1 pancreatic tumor model, in which the hRS7 (anti-EGP-1), hPAM4 (anti-mucin) and hMN-14 (anti-CEACAM5) conjugates CL2A-SN-38 showed better efficacy than the hA20-CL2A-SN-38 conjugated control (anti-CD20) and untreated control. Similarly, in a BXPC3 model of human pancreatic cancer, the specific hRS7-CL2A-SN-38 showed better therapeutic efficacy than control treatments (F igure 3). Likewise, in an aggressive LS174T model of human colon carcinoma, treatment with specific hMN-14-CL2A-SN-38 was more effective than no treatment (F igure 4).

E x a m p le 12. The u se of an tic ue rp oan ti-T ROP -2 Ig G -SN -38 w ith ju gado for the effi cient treatment zded iv e rso sc á nce re sep ite lia I know

[0265 ] The purpose of this study was to evaluate the efficacy of an SN-38-anti-TROP-2 antibody-drug conjugate (ADC) against various types of human solid tumors, and to assess its tolerability in mice and monkeys, these the latter with tissue cross-reactivity to hRS7 similar to humans. Two SN-38 derivatives, CL2-SN-38 (see US Patent No. 7,591,994) and CL2ASN-38, were conjugated to the humanized anti-TROP-2 antibody, hRS7. Immunoconjugates were characterized in vitro for stability, binding, and cytotoxicity. Efficacy was tested in five different human solid tumor xenograft models expressing TROP-2 antigen. Toxicity was evaluated in mice and Cynomolgus monkeys.

[0266 ] The hRS7 conjugates of the two SN-38 derivatives were equivalent in drug substitution (~6 ), cell binding ( Kd ~ 1.2 nmol/L), cytotoxicity (IC50 ~ 2.2 nmol/L) , and in vitro serum stability (t/k ~ 20 hours). Exposure of cells to ADC demonstrated signaling pathways leading to PARP cleavage, but differences versus free SN-38 were observed in upregulation of p53 and p21. Significant antitumor effects were produced with hRS7-SN-38 at non-toxic doses in mice bearing Calu-3 (P < 0.05), Capan-1 (P < 0.018), BxPC-3 (P < 0.005) and COLO tumors. 205 (P < 0.033) compared to untargeted control ADCs. Mice tolerated a dose of 2 x 12 mg/kg (SN-38 equivalents) with only short-lived elevations in ALT and AST liver enzyme levels. Cynomolgus monkeys infused with 2 x 0.96 mg/kg exhibited only transient decreases in blood counts, although, more importantly, values did not fall below normal ranges.

[0267 ] It is concluded that the anti-TROP-2 hRS7-CL2A-SN-38 ADC provides significant and specific antitumor effects against a range of human solid tumor types. It was well tolerated in monkeys, with tissue TROP-2 expression similar to that in humans. (Cardillo et al, 2011, Cancer Clin Res. 17: 3157-69)

[0268 ] Successful treatment of irinotecan in patients with solid tumors has been limited due in large part to the low rate of conversion of the prodrug of CPT-11 to the active Metabolite SN-38. Others have examined non-targeted forms of SN-38 as a means of circumventing the need for this conversion and passively delivering SN-38 to tumors. We conjugated SN-38 covalently to a humanized anti-TROP-2 antibody, hRS7. This antibody-drug conjugate has specific antitumor effects in a variety of sc human cancer xenograft models, including non-small cell lung carcinoma, pancreatic, colorectal, and squamous cell lung carcinomas, all at non-toxic doses (eg, ., < 3.2 mg/kg cumulative equivalent dose of SN-38).

[0269 ] TROP-2 is widely expressed in many epithelial cancers, but also some normal tissues, and therefore a dose escalation study in cynomolgus monkeys was performed to assess the clinical safety of this conjugate. Monkeys tolerated 24 mg SN-38 equivalents/kg with minor and reversible toxicities. Given its tumor-targeting and safety profile, hRS7-CL2A-SN-38 may provide an improvement in the treatment of irinotecan-responsive solid tumors.

[0270 ] Human trophoblast cell surface antigen (TROP-2), also known as GA733-1 (gastric antigen 733-1), EGP-1 (epithelial glycoprotein-1), and TACSTD2 (calcium-associated signal transducer tumors), is expressed in a variety of human carcinomas and is of prognostic significance in some, being associated with more aggressive disease (see, e.g., Alberti et al., 1992, Hybridoma 11:539-45; Stein et al ., 1993, Int J Cancer 55:938-46; Stein et al., 1994, Int J Cancer Suppl. 8:98-102). Studies of the functional role of TROP-2 in a mouse pancreatic cancer cell line transfected with murine TROP-2 revealed increased proliferation under low serum conditions, anchorage-independent growth and migration in vitro, and an increased growth rate. enhanced with evidence of increased expression of Ki-67 in vivo and a higher probability of metastasizing (Cubas et al., 2010, Mol Cancer 9: 253).

[0271] The distribution of the TROP-2 antigen in many epithelial cancers makes it an attractive therapeutic target. Stein et al. (1993, Int J Cancer 55: 938-46) characterized an antibody, designated RS7-3G11 (RS7), that bound EGP-1, which was present on several solid tumors, but the antigen was also expressed on some normal tissues. , usually at a lower intensity or in restricted regions. Therapeutic and targeting efficacies in various human tumor xenografts using radiolabeled RS7 were documented (Shih et al., 1995, Cancer Res 55:5857s-63s; Stein et al., 1997, Cancer 80:2636-41; Govindan et al. ., 2004, Breast Cancer Res Treat 84: 173-82), but this internalizing antibody did not show therapeutic activity in unconjugated form (Shih et al., 1995, Cancer Res 55: 5857s-63s). However, in vitro it has demonstrated antibody-dependent cellular cytotoxicity (ADCC) activity against TROP-2 positive carcinomas.

[0272] We have reported the preparation of antibody-drug conjugates (ADC), using an anti-CEACAM5 (CD66e) IgG coupled to various derivatives of SN-38, a topoisomerase-I inhibitor that is the active component of irinotecan or CpT -11 (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res 15:6052-61). The derivatives varied in their in vitro serum stability properties, and in vivo studies found that one form (termed CL2) is more effective in preventing or arresting the growth of human colon and pancreatic cancer xenografts than other linkages. more or less stability.

[0273] Importantly, these effects occurred at non-toxic doses, and initial testing failed to determine dose-limiting toxicity (Govindan et al., 2009, Clin Cancer Res 15:6052-61). These results were encouraging, but also surprising, because the CEACAM5 antibody is not internalized, a property thought to be critical to the success of an ADC. We speculated that the therapeutic activity of the anti-CEACAM5-SN-38 conjugate might be related to the slow release of SN-38 within the tumor after the antibody localized. Because irinotecan works best when cells are exposed during the S phase of their growth cycle, sustained release is expected to improve responses. In fact, SN-38 coupled to non-targeted plasma extenders, such as polyethylene glycol (PEG) or micelles, has shown improved efficacy over irinotecan or SN-38 alone (eg, Koizumi et al., 2006, Cancer Res 66 : 10048-56), giving additional support to this mechanism.

[0274] Given the broad reactivity of the RS7 antibody with epithelial cancers and its ability to internalize, we hypothesized that an RS7-SN-38 conjugate could benefit not only from sustained drug release, but also from direct intracellular delivery. Therefore, we prepared and tested the efficacy of SN-38 conjugates using a humanized version of the murine RS7 (hRS7) antibody. A modification was made to the SN-38 derivative (Govindan et al., 2009, Clin Cancer Res 15: 6052-61), which improved the quality of the conjugate without altering its in vitro stability or in vivo efficacy. This new derivative (designated CL2A) is currently the preferred agent for coupling SN-38 to antibodies. Here, we show the efficacy of the hRS7-SN-38 conjugate on various epithelial cancer cell lines implanted in nude mice at non-toxic doses, with further studies revealing that substantially higher doses could be tolerated. More importantly, toxicity studies in monkeys also expressing TROP-2 in human-like tissues showed that hRS7-CL2A-SN-38 was tolerated at amounts appreciably higher than the therapeutically effective dose in mice, providing evidence that this conjugate is a promising agent for the treatment of patients with a wide range of epithelial cancers.

Materials and methods

[0275] Cell lines, antibodies and chemotherapeutics. All human cancer cell lines used in this study were purchased from the American Type Culture Collection. These include Calu-3 (non-small cell lung carcinoma), SK-MES-1 (squamous cell lung carcinoma), COLO 205 (colon adenocarcinoma), Capan-1 and BxPC-3 (pancreatic adenocarcinomas), and PC-3 (prostate adenocarcinomas). Humanized RS7 IgG and control humanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22 (hLL2 IgG, epratuzumab) antibodies were prepared by Immunomedics, Inc. Irinotecan (20 mg/mL) was obtained from Hospira, Inc.

[0276] SN-38 immunoconjugates and in vitro aspects. The synthesis of CL2-SN-38 has been previously described (Moon et al., 2008, J Med Chem 51:6916-26, see also US Patent No. 7,591,994). Its conjugation with hRS7 IgG and serum stability were performed as described (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res 15:6052-61). Preparations of CL2A-SN-38 (MW 1480) and its hRS7 conjugate, and stability, binding, and cytotoxicity studies, were performed as previously described (Moon et al., 2008, J Med Chem 51:6916-26). Cell lysates were prepared and immunoblotted for p21 Waf1/Cip, p53 and PARP (poly-ADP-ribose polymerase).

[0277] In vivo therapeutic studies . For all animal studies, doses of SN-38 and irinotecan immunoconjugates are shown in SN-38 equivalents. Based on a mean SN-38/IgG substitution ratio of 6, a 500 pg dose of ADC for a 20 g mouse (25 mg/kg) contains 0.4 mg/kg SN-38. Irinotecan doses are also shown as SN-38 equivalents (ie, 40 mg irinotecan/kg is equivalent to 24 mg/kg SN-38). Female NCr ( nu/nu) athymic nude mice, 4 to 8 weeks old, and male SwissWebster mice, 10 weeks old, were purchased. weeks old, from Taconic Farms. Tolerability studies were performed in Cynomolgus monkeys ( Macaca fascicularis; 2.5-4 kg male and female) by SNBL USA, Ltd. The animals were implanted subcutaneously with different human cancer cell lines. Tumor volume (TV) was determined by 2-dimensional measurements using calipers, with volumes defined as: L x w2/2, where L is the longest dimension of the tumor and w is the shortest. The tumors were between 0.10 and 0.47 cm3 in size when therapy began. Treatment regimens, doses, and number of animals in each experiment are described in the Results. Lyophilized hRS7-CL2A-SN-38 and control ADC were reconstituted and diluted as required in sterile saline. All reagents were administered intraperitoneally (0.1 ml), except irinotecan, which was administered intravenously. The dosing regimen was influenced by our previous investigations, where the ADC was administered every 4 days or twice a week for varying periods of time (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al. , 2009, Clin Cancer Res 15: 6052-61). This dosing frequency reflected a consideration of the serum half-life of the conjugate in vitro, to allow for more continuous exposure to ADC.

[0278] Statistics. Growth curves were determined as percent change in initial VT over time. Statistical analysis of tumor growth was based on the area under the curve (AUC). Growth profiles of individual tumors were obtained by linear curve modeling. An f test was used to determine equality of variance between groups before statistical analysis of growth curves. A 2-tailed t- test was used to assess statistical significance between the various treatment groups and controls, except for the saline control, where a 1-tailed t- test was used (significance at P < 0.05). Statistical comparisons of AUC were made only up to the time that the first animal within a group was euthanized due to progression.

[0279] Pharmacokinetics and biodistribution. 111In-radiolabeled hRS7-CL2A-SN-38 and hRS7 IgG were injected into nude mice bearing sc SK-MES-1 tumors (~0.3 cm 3 ). One group was injected intravenously with 20 jC i (250 pg of protein) of 111In-hRS7-CL2A-SN-38, while another group received 20 jC i (250 pg of protein) of 111In-hRS7 IgG. At various time points, mice (5 per time point) were anesthetized, bled by intracardiac puncture, and then sacrificed. Tumors and various tissues were removed, weighed and counted by scintillation and to determine the percent injected dose per gram of tissue (%Dl/g). A third group was injected with 250 μg of unlabeled hRS7-CL2A-SN-38 3 days before 111In-hRS7-CL2A-SN-38 administration and was also autopsied. A 2-tailed t- test was used to compare hRS7-CL2A-SN-38 and hRS7 IgG uptake after determining equality of variance using the f-test. Pharmacokinetic analysis of blood clearance was performed using WinNonLin software (Parsight Corp.).

[0280] Tolerability in Swiss-Webster mice and Cynomolgus monkeys. Briefly, mice were sorted into 4 groups each to receive 2 mL ip injections of a sodium acetate buffer control or 3 different doses of hRS7-CL2A-SN-38 (4.8 or 12 mg/kg s N-38) on days 0 and 3 followed by blood and serum collection, as described in Results. Cynomolgus monkeys (3 males and 3 females; 2.5-4.0 kg) were administered 2 different doses of hRS7-CL2A-SN-38. Doses, times, and number of monkeys bled to assess potential hematologic toxicities and serum chemistry are described in the Results.

Results

[0281] Stability and potency of hRS7-CL2A-SN-38. Two different ligands were used to conjugate SN-38 to hRS7 IgG. The first is designated CL2-SN-38 and has been previously described (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res 15:6052-61). A change was made in the CL2 linker synthesis in that the phenylalanine moiety was removed. This change simplified the synthesis, but did not affect the conjugation result (eg, both CL2-SN-38 and CL2A-SN-38 incorporated ~6 SN-38 per IgG molecule). Side-by-side comparisons found no significant differences in serum stability, antigen binding, or in vitro cytotoxicity (not shown).

[0282] To confirm that changing the SN-38 linker from CL2 to CL2A did not impact potency in vivo, hRS7-CL2A and hRS7-CL2-SN-38 were compared in mice bearing COLO 205 or Capan 1 tumors (not shown), using SN-38 0.4 mg or 0.2 mg/kg twice weekly for 3 to 4 weeks, respectively, and with initial tumors of 0.25 cm3 in size in both studies. Both hRS7-CL2A and CL2-SN-38 conjugates significantly inhibited tumor growth compared to (AUC14days P<0.002 vs. saline in the COLO 205 model; AUC21days P<0.001 vs. saline in the Capan-1 model) untreated, and a non-target anti-CD20 control ADC, hA20-CL2A-SN-38 (AUC14 days P<0.003 in COLO-205 model; at Uc 35 days: P<0.002 in Capan-1 model). At the end of the study (day 140) in the Capan-1 model, 50% of hRS7-CL2A-SN-38 treated mice and 40% of hRS7-CL2-SN-38 mice were tumor-free, while that only 20% of animals treated with hA20-ADC had no visible signs of disease. Importantly, there was no difference in efficacy between the 2 specific conjugates in both tumor models.

[0283] Mechanism of action. In vitro cytotoxicity studies demonstrated that hRS7-CL2A-SN-38 had IC50 values in the nmol/L range against several different solid tumor lines (Table 9). The IC50 with SN-38 connection was lower than the conjugate in all cell lines. Although there was no correlation between TROP-2 expression and sensitivity to hRS7-CL2A-SN-38, the IC50 ratio of ADC versus free SN-38 was lower in cells expressing more TROP-2, which probably reflects the increased ability to internalize the drug when more antigen is present.

T ab le 9. E xp re s io n o f TROP -2 and in vitro cytot o x ic ity of SN -38 and h RS 7 -SN -38 in various lines of solid tu m o re s

TROP-2 Expression via FACS Cytotoxicity Results Line Fluorescence Percent SN-38 95% CI hRS7- 95% CI Median Positive Cell Ratio SN-38a ADC/SN- (background) IC50 IC50 IC50 IC50 38 free (nmol/L ) (nmol/L) (nmol/L) (nmol/L)

Calu-3 282.2 (4.7) 99.6% 7.19 5.77-8.95 9.97 8 .12 1.39 12.25

COLO 141.5 (4.5) 99.5% 1.02 0.66-1.57 1.95 1.26-3.01 1.91

205

Capan-1 100.0 (5.0) 94.2% 3.50 2.17-5.65 6.99 5.02-9.72 2.00

PC-3 46.2 (5.5) 73.6% 1.86 1.16-2.99 4.24 2.99-6.01 2.28

SK-MES- 44.0 (3.5) 91.2% 8.61 6.30 23.14 17.98 2.69

1 11.76 29.78

BxPC-3 26.4 (3.1) 98.3% 1.44 1.04-2.00 4.03 3.25-4.98 2.80 aIC50 value is shown as SN-38 equivalents of hRS7- SN-38

[0284 ] SN-38 is known to activate various signaling pathways in cells, leading to apoptosis. Our initial studies examined the expression of 2 proteins involved in early signaling events (p21 WAF1/Cip1 and p53) and 1 late apoptotic event [poly-ADP-ribose polymerase (PARP) cleavage] in vitro (not shown). In BxPC-3, sN-38 led to a 20-fold increase in p21 WAF1/Cip1 expression, whereas hRS7-CL2A-SN-38 resulted in only a 10-fold increase, a finding consistent with the higher activity. with SN-38 in this cell line (T ab la 9). However, hRS7-CL2A-SN-38 increased p21 WAF1/Cip1 expression in Calu-3 more than 2-fold over free N-38 (not shown).

[0285 ] A greater disparity between hRS7-CL2A-SN-38- and free SN-38 mediated signaling events was observed in p53 expression. In both BxPC-3 and Calu-3, p53 upregulation with free SN-38 was not evident until 48 hours, whereas hRS7-CL2A-SN-38 upregulated p53 at 24 hours (not shown). . Furthermore, p53 expression in cells exposed to ADC was higher in both cell lines compared to SN-38 (not shown). Interestingly, although hRS7 IgG had no appreciable effect on p21WAF1/Cip1 expression, it induced p53 upregulation in both BxPC-3 and Calu-3, but only after 48 hours exposure. In terms of subsequent apoptotic events, PARP cleavage was evident in both cell lines when incubated with SN-38 or the conjugate (not shown). The presence of the cleaved PARP was higher at 24 hours in BxPC-3, which correlates with the high expression of p21 and its lower IC50. The higher degree of cleavage with free SN-38 on the ADC was consistent with the cytotoxicity findings.

[0286] Efficacy of hRS7-SN-38. Because TROP-2 is widely expressed in a number of human carcinomas, studies were performed in a number of different human cancer models, beginning with an evaluation of the hRS7-CL2-SN-38 linkage, but later using conjugates with the CL2A linkage. Calu-3-bearing nude mice receiving 0.04 mg SN-38/kg hRS7-CL2-SN-38 every 4 days 3 4 had a significantly improved response compared to animals receiving the equivalent amount of hLL2 -CL2-SN-38 (VT = 0.14 ± 0.22 cm3 vs. 0.80 ± 0.91 cm3, respectively; AUC 42 days P<0.026; FIG. 5A). A dose response was observed when the dose was increased to 0.4 mg/kg SN-38. At this higher dose level, all mice receiving the specific hRS7 conjugate were "cured" by 28 days and remained tumor-free until the end of the study on day 147, whereas tumors regrown in treated animals with ADC irrelevant (specific vs. AUC irrelevant 98 days: P = 0.05). In mice receiving the mixture of hRS7 IgG and SN-38, tumors progressed >4.5 times per day 56 (VT = 1.1060.88 cm3; AUC 56 days P<0.006 vs. hRS7-CL2-SN- 38).

[0287 ] Efficacy was also examined in human colonic (COLO 205) and pancreatic (Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (FIG. 5B), hRS7-CL2-SN-38 (0.4 mg/kg, q4dx8) prevented tumor growth during the 28-day treatment period with significantly smaller tumors in comparison with anti-CD20 ADC control. (hA20-CL2-SN-38), or hRS7 IgG (VT = 0.16 ± 0.09 cm3, 1.19 ± 0.59 cm3, and 1.77 ± 0.93 cm3, respectively; AUC28 days P<0.016 ). Irinotecan MTD (24 mg SN-38/kg, q2dx5) was as effective as hRS7-CL2-SN-38, because mouse serum can convert irinotecan to SN-38 more efficiently than human serum, but the dose of SN-38 in irinotecan (2,400 pg cumulative) was 37.5-fold higher than in the conjugate (64 pg total).

[0288 ] Animals bearing Capan-1 did not show a significant response to irinotecan alone when given a dose of SN-38 equivalent to the hRS7-CL2-SN-38 conjugate (eg, on day 35, the size mean tumor was 0.04 ± 0.05 cm3 in animals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78 ± 0.62 cm3 in irinotecan-treated animals given 0, 4 mg/kg SN-38, AUC day 35 P<0.001, FIG 5C). When the dose of irinotecan was increased 10-fold to 4 mg/kg SN-38, the response improved, but was still not as significant as the conjugate at the dose level of irinotecan. SN-38 0.4 mg/kg (VT = 0.17 ± 0.18 cm3 vs. 1.69 ± 0.47 cm3, AUC d¡a49 P<0.001). An equal dose of untargeted hA20-CL2-SN-38 also had a significant antitumor effect compared to irinotecan-treated animals, but the hRS7-specific conjugate was significantly better than the irrelevant ADC (VT = 0.17 ± 0, 18 cm3 vs. 0.80 ± 0.68 cm3, AUCday49 P<0.018).

[0289] Studies with the hRS7-CL2A-SN-38 ADC were then extended to 2 other models of human epithelial cancers. In mice bearing BxPC-3 human pancreatic tumors (FIG. 5D), hRS7-CL2A-SN-38 again significantly inhibited tumor growth compared to control mice treated with saline or an equivalent amount of hA20-CL2A-SN. -38 untargeted (VT = 0.24 ± 0.11 cm3 vs. 1.17 ± 0.45 cm3 and 1.05 ± 0.73 cm3, respectively; AUC day21 P<0.001), or irinotecan administered at a dose 10-fold higher SN-38 equivalent (VT = 0.27 ± 0.18 cm3 vs. 0.90 ± 0.62 cm3, respectively; AUC day25 P<0.004). Interestingly, in mice bearing SK-MES-1 human squamous cell lung tumors treated with 0.4 mg/kg ADC (FIG. 5E), tumor growth inhibition was superior to saline or unconjugated hRS7 IgG. (VT = 0.36 ± 0.25 cm3 vs 1.02 ± 0.70 cm3 and 1.30 ± 1.08 cm3, respectively; AUC28 days, P<0.043), but hA20-CL2A-SN-38 did not target or the MTD of irinotecan provided the same antitumor effects as the specific hRS7-SN-38 conjugate. In all murine studies, the hRS7-SN-38 ADC was well tolerated in terms of body weight loss (not shown).

[0290] Biodistribution of hRS7-CL2A-SN-38. Biodistributions of hRS7-CL2A-SN-38 or unconjugated hRS7 IgG in mice bearing SK-MES-1 human lung squamous cell carcinoma xenografts (not shown) were compared using the respective 111 In-labeled substrates. Pharmacokinetic analysis was performed to determine the clearance of hRS7-CL2A-SN-38 relative to unconjugated hRS7 (not shown). ADC cleared faster than the equivalent amount of unconjugated hRS7, with ADC exhibiting ~40% shorter half-life and mean residence time. However, this had minimal impact on tumor uptake (not shown). Although there were significant differences at the 24 and 48 hour time points, at 72 hours (peak uptake) the amounts of both agents in the tumor were similar. Among normal tissues, hepatic and splenic differences were the most striking (not shown). At 24 hours post-injection, there was >2-fold more hRS7-CL2ASN-38 in the liver than hRS7 IgG. In contrast, 3-fold more parental hRS7 IgG was present in the spleen at maximal uptake (48 hour time point) than hRS7-CL2A-SN-38. Uptake and clearance in other tissues generally reflect differences in blood concentration.

[0291] Since twice-weekly doses were given for therapy, tumor uptake in a group of animals that first received a predose of 0.2 mg/kg (250 µg of protein) of hRS7 ADC 3 days before injection 111 In-labeled antibody was examined. Tumor uptake of 111In-hRS7-CL2A-SN-38 in predosed mice was substantially reduced at each time point compared to animals that did not receive predose (eg, at 72 hours, predosed tumor uptake was 12.5% ± 3.8% Dl/g vs. 25.4% ± 8.1% Dl/g in naïve animals, P = 0.0123). Predosing had no appreciable impact on blood clearance or tissue absorption (not shown). These studies suggest that in some tumor models, tumor accumulation of specific antibody may be reduced by the above doses, which probably explains why the specificity of a therapeutic response might decrease with increasing doses of ADC and why it is not indicated. possible dose increase.

[0292] Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice and Cynomolgus monkeys. Swiss-Webster mice tolerated 2 doses for 3 days, each of 4, 8 and 12 mg SN-38/kg hRS7-CL2A-SN-38, with minimal transient weight loss (not shown). Hematopoietic toxicity did not occur, and serum chemistries revealed only elevated aspartate transaminase (AST) and alanine transaminase (not shown). Seven days after treatment, AST was elevated above normal levels (>298 U/L) in all 3 treatment groups (not shown), with the highest proportion of mice in the 2 x 8 mg/L group. kg. However, at 15 days after treatment, most of the animals were within the normal range. ALT levels were also above the normal range (>77 U/L) within 7 days of treatment (not shown) and with evidence of normalization by day 15. The livers of all these mice showed no histological evidence of ALT levels. tissue damage (not shown). In terms of kidney function, only glucose and chloride levels were somewhat elevated in the treated groups. At 2 x 8 mg/kg, 5 of 7 mice had mildly elevated glucose levels (range 273-320 mg/dL, upper limit of normal 263 mg/dL) that returned to normal 15 days after injection. Similarly, chloride levels were slightly elevated, ranging from 116 to 127 mmol/L (upper limit of normal range 115 mmol/L) in the 2 highest dose groups (57% in the 2 x 8 mg group). /kg and 100% of the mice in the 2 x 12 mg/kg group), and remained elevated up to 15 days after injection. This could also be indicative of gastrointestinal toxicity, because most of the chloride is obtained by absorption in the intestine; however, at completion, there was no histologic evidence of tissue damage in any organ system examined (not shown).

[0293] Because mice do not express TROP-2 bound by hRS7, a more suitable model was required to determine the potential of the hRS7 conjugate for clinical use. Immunohistology studies revealed binding in multiple tissues in both humans and Cynomolgus monkeys (breast, eye, gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas, parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsils). , ureter, urinary bladder, and uterus; not shown). Based on this cross-reactivity, a tolerability study in monkeys was performed.

[0294] The group receiving 2 x 0.96 mg SN-38/kg hRS7-CL2A-SN-38 had no significant clinical events after infusion and through completion of the study. Weight loss did not exceed 7.3% and returned to acclimatization weights by day 15. Transient decreases were observed in most blood count data (not shown), but values did not fall below normal ranges. normal. No abnormal values were found in serum chemistry. The histopathology of the animals necropsied on day 11 (8 days after the last injection) showed microscopic changes in the hematopoietic organs (thymus, mandibular and mesenteric lymph nodes, spleen and bone marrow), gastrointestinal organs (stomach , duodenum, jejunum, ileum, cecum, colon, and rectum), the female reproductive organs (ovary, uterus, and vagina), and at the injection site. These changes ranged from minimal to moderate and were completely reversed by the end of the recovery period (day 32) in all tissues except the thymus and gastrointestinal tract, which tended toward full recovery at the latter time point.

[0295] At the dose level of 2 x 1.92 mg SN-38/kg conjugate, there was 1 death from gastrointestinal complications and bone marrow suppression, and other animals within this group showed similar adverse events, but more severe than the 2 x 0.96 mg/kg group. These data indicate that the dose-limiting toxicities were identical to those of irinotecan; namely, intestinal and hematologic. Therefore, the MTD for hRS7-CL2A-SN-38 is between 2 x 0.96 and 1.92 mg SN-38/kg, which represents a human equivalent dose of 2 x 0.3 to 0.6 mg /kg SN-38.

Discussion

[0296] TROP -2 is a protein expressed in many epithelial tumors, including lung, breast, colorectal, pancreatic, prostate, and ovarian cancers, making it a potentially important target for delivering cytotoxic agents. The RS7 antibody is internalized when it binds to TROP-2 (Shih et al., 1995, Cancer Res 55:5857s-63s), allowing direct intracellular delivery of cytotoxics.

[0297] The conjugation of chemotherapeutic drugs with antibodies has been explored for more than 30 years. Because a substantial part of an ADC is not processed by the tumor, but by normal tissues, there is a risk that these agents will be too toxic to normal organ systems before reaching therapeutic level in tumors. As with any therapeutic, the therapeutic window is a key factor determining the potential of an ADC, and therefore, instead of examining "ultratoxic" drugs, we chose SN-38 as the pharmacological component of the TROP-targeting ADC. two.

[0298] SN-38 is a potent inhibitor of topoisomerase-I, with IC50 values in the nanomolar range in various cell lines. It is the active form of the prodrug, irinotecan, which is used to treat colorectal cancer and is also active in lung, breast, and brain cancers. We reasoned that a directly targeted SN-38, in the form of ADCs, would be a significantly improved therapeutic over CPT-11, by overcoming the latter's low and variable patient bioconversion to active SN-38.

[0299] The Phe-Lys peptide inserted into the original CL2 derivative allowed for possible cleavage by cathepsin B. In an effort to simplify the synthesis process, in CL2A, phenylalanine was removed, and thus the site of Cathepsin B cleavage. Interestingly, this product had a better defined chromatographic profile compared to the broad profile obtained with CL2 (not shown), but more importantly, this change had no impact on binding, stability, or potency. of the conjugate in parallel tests. These data suggest that SN-38 in CL2 was released from the conjugate primarily by cleavage at the pH-sensitive benzyl carbonate bond to the lactone ring of SN-38 and not by the cathepsin B cleavage site.

[0300] The in vitro cytotoxicity of hRS7 ADC against a number of solid tumor cell lines consistently had IC50 values in the nmol/L range. However, cells exposed to free SN-38 demonstrated a lower IC50 value compared to ADC. This disparity between free and conjugated SN-38 was also reported for ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96) and NK012 (Koizumi et al., 2006, Cancer Res 66:10048-56). ). ENZ-2208 uses a branched PEG to bind about 3.5 to 4 molecules of SN-38 per PEG, while NK012 is a micellar nanoparticle containing 20% SN-38 by weight. With our ADC, this disparity (i.e., the potency ratio with free vs. conjugated SN-38) decreased as TROP-2 expression levels in tumor cells increased, suggesting an advantage for targeted delivery. of the drug. In terms of in vitro serum stability, the CL2 and CL2A-SN-38 forms of hRS7-SN-38 produced a t/A of ~20 hours, which is in contrast to the short tPA of 12.3 minutes reported for ENZ-2208 ( Zhao et al., 2008, Bioconjug Chem 19: 849-59), but similar to the 57% release of SN-38 from NK012 under physiological conditions after 24 hours (Koizumi et al., 2006, Cancer Res 66: 10048- 56).

[0301] Treatment of tumor-bearing mice with hRS7-SN-38 (with either CL2-SN-38 or CL2A-SN-38) significantly inhibited tumor growth in 5 different tumor models. In 4 of them, tumor regressions were observed, and in the case of Calu 3, all mice that received the highest dose of hRS7-SN-38 were free of tumors at the end of the study. Unlike in humans, irinotecan is highly efficiently converted to SN-38 by a plasma esterase in mice, with a conversion rate greater than 50%, and produces greater efficacy in mice than in humans. When irinotecan was given at 10-fold or equivalent levels of SN-38, hRS7-SN-38 was significantly better at controlling tumor growth. Only when irinotecan was administered at its DMT of 24 mg/kg q2dx5 (37.5-fold more SN-38) did it match the efficacy of hRS7-SN-38. In patients, we would expect this advantage to further favor hRS7-CL2A-SN-38, because the bioconversion of irinotecan would be substantially less.

[0302] It also showed in some cell lines expressing the antigen, such as SK-MES-1, that the use of an antigen-binding ADC does not guarantee a better therapeutic response than an irrelevant non-binding conjugate. This is not an unusual or unexpected finding. In fact, the aforementioned non-binding SN-38 conjugates improve therapeutic activity compared to irinotecan, so an irrelevant IgG-SN-38 conjugate is expected to have some activity. This is related to the fact that tumors have immature and leaky vessels that allow the passage of macromolecules better than normal tissues. With our conjugate, 50% of the SN-38 will be released in ~13 hours when the pH is lowered to a level that mimics lysosomal levels (eg, pH5.3 at 37°C; data not shown), while that in the neutral pH of whey, the release rate is almost doubled. If an irrelevant conjugate enters an acidic tumor microenvironment, it is expected to release some SN-38 locally. Other factors, such as tumor physiology and innate drug sensitivities, will also play a role in defining this baseline activity. However, a specific conjugate with a longer residence time should have improved potency over this initial response as long as there is enough antigen to capture the specific antibody. Biodistribution studies in the SK-MES-1 model also showed that if the tumor antigen becomes saturated as a consequence of successive dosing, tumor uptake of the specific conjugate is reduced, producing therapeutic results similar to those found with an irrelevant conjugate. .

[0303] Although it is difficult to make direct comparisons between our ADC and published reports of other SN-38 delivery agents, some general observations can be made. In our therapy studies, the highest single dose was 0.4 mg/kg SN-38. In the Calu-3 model, only 4 injections were given for a total cumulative dose of 1.6 mg/kg SN-38 or 32 pg SN-38 in a 20 g mouse. Multiple studies were conducted with ENZ-2208 using its MTD of 10 mg/kg x 5, and preclinical studies with NK012 involved its MTD of 30 mg/kg x 3. Thus, significant antitumor effects were obtained with hRS7-SN- 38- to 30-fold and 55-fold fewer SN-38 equivalents than the doses reported in ENZ-2208 and NK012, respectively. Even with 10-fold less hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed, while lower doses of ENZ-2208 did not occur, and when the dose of NK012 was reduced 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al., 2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity at a 1-week cumulative dose of 24 mg/kg SN-38 (1,500 mg/kg conjugate), indicating that the MTD was higher. Thus, tumor-bearing animals were effectively treated with 7.5- to 15-fold lower amounts of SN-38 equivalents.

[0304] As an inhibitor of topoisomerase-I, SN-38 induces significant damage to a cell's DNA, with upregulation of p53 and p21 WAF1/Cip1 resulting in caspase activation and PARP cleavage. When we exposed BxPC-3 and Calu-3 cells to our ADC, both p53 and p21 WAF1/Cip1 were upregulated above basal levels. Furthermore, PARP cleavage was also evident in both cell lines, confirming an apoptotic event in these cells. Of interest was the increased upregulation of p21 WAF1/Cip1 in BxPC-3 and Calu-3 relative to p53 by both free SN-38 and our hRS7-SN-38. This may be indicative of the mutational status of p53 in these 2 cell lines and the use of a p53-independent pathway for p21 WAF1/Cip1-mediated apoptosis.

[0305] An interesting observation was the first upregulation of p53 in BxPC-3 and Calu-3 at 24 hours mediated by hRS7-ADC relative to free SN-38. Even naked hRS7 IgG could upregulate p53 in these cell lines, albeit only after a 48-hour exposure. TROP-2 overexpression and cross-linking by antibodies has been linked to several MAPK-related signaling events, as well as intracellular calcium release. While hRS7 binding was not sufficient to induce apoptosis in BxPC-3 and Calu-3, as evidenced by the lack of PARP cleavage, it may be sufficient to prime a cell, so inclusion of conjugated SN-38 with hRS7 can lead to a greater effect on the inhibition of tumor growth. Studies are currently underway to understand which pathways are involved with hRS7 delivery of SN-38 and how they may differ from free SN-38, and what effect p53 status may have on this signaling.

[0306] Biodistribution studies revealed that hRS7-CL2A-SN-38 had similar tumor uptake as parental hRS7 IgG, but was cleared substantially faster with 2-fold higher hepatic uptake, which may be due to hydrophobicity of SN-38. With the elimination of ADC through the liver, hepatic and gastrointestinal toxicities were expected to be dose limiting. Although the mice had evidence of increased hepatic transaminases, gastrointestinal toxicity was mild at best, with only transient weight loss, and no abnormalities were noted on histopathologic examination. Interestingly, no hematological toxicity was observed. However, monkeys showed a toxicity profile identical to that expected for irinotecan, with gastrointestinal and haematological toxicity being dose-limiting.

[0307] Because TROP-2 recognized by hRS7 is not expressed in mice, it was critically important to carry out toxicity studies in monkeys that have similar tissue expression of TROP-2 as humans. Monkeys tolerated 0.96 mg/kg/dose (~12 mg/m2) with mild and reversible toxicity, which extrapolates to a human dose of ~0.3 mg/kg/dose (~11 mg/m2). In a phase I clinical trial of NK012, patients with solid tumors tolerated SN-38 28 mg/m2 every 3 weeks with grade 4 neutropenia as a dose-limiting toxicity (Hamaguchi et al., 2010, Clin Cancer Res 16 : 5058-66). Similarly, phase I clinical trials with ENZ-2208 revealed dose-limiting febrile neutropenia, with the recommendation to administer 10 mg/m2 every 3 weeks or 16 mg/m2 if patients received G-CSF. Because monkeys tolerated a cumulative human equivalent dose of 22 mg/m2, it is possible that although hRS7 binds to various normal tissues, the MTD for a single treatment of hRS7 ADC could be similar to that of other non-target SN-38 agents. In fact, the specificity of the anti-TROP-2 antibody did not appear to play a role in defining DLT, because the toxicity profile was similar to that of irinotecan. More importantly, if antitumor activity can be achieved in humans as in responding mice with a human equivalent dose of only 0.03 mg SN-38 equivalents/kg/dose, then clinically significant antitumor responses could be elicited.

[0308] In conclusion, toxicology studies in monkeys, combined with in vivo human cancer xenograft models in mice have indicated that this TROP-2 targeting ADC is an effective therapeutic in various tumors of different epithelial origin.

Example 13. SN-38 conjugated by anti-CD22 (epratuzumab) for the therapy of hematological disorders

[0309] We have previously found that SN-38-conjugated slow internalizing antibodies could be used successfully when prepared with a CL2A linker that allows approximately 50% of IgG-bound SN-38 to dissociate in serum every 24 hours. In this study, the efficacy of SN-38 conjugates prepared with epratuzumab (rapid internalization) and veltuzumab (slow internalization), humanized anti-CD22 and anti-CD20 IgG, respectively, for the treatment of B-cell malignancies was examined. Both antibody-drug conjugates had similar nanomolar activity against a variety of human lymphoma/leukemia cell lines, but the slow release of SN-38 compromised in vitro potency discrimination even against an irrelevant conjugate. When SN-38 was stably bound to the anti-CD22 conjugate, its potency was reduced 40- to 55-fold. Therefore, further studies were performed only with the less stable and slow dissociating CL2A linker. In vivo, similar antitumor activity was found between CD22 and CD20 antibody-drug conjugate in mice bearing Ramos xenografts, although Ramos expressed 15-fold more CD20 than CD22, suggesting that internalization of the epratuzumab-SN-38 conjugate ( EmabSN-38) reinforced its activity. Emab-SN-38 was more effective than an irrelevant and non-binding IgG-SN-38 conjugate in vivo, killing most well-established Ramos xenografts at non-toxic doses. In vitro and in vivo studies showed that Emab-CL2A-SN-38 could be combined with unconjugated veltuzumab for more effective treatment. Thus, EmabSN-38 is active in lymphoma and leukemia at doses well below toxic levels and thus represents a promising new agent with therapeutic potential alone or in combination with anti-CD20 antibody therapy. (Sharkey et al., 2011, Mol Cancer Ther 11:224-34.)

Introduction

[0310] Significant effort has focused on biologic therapy of leukemia and lymphoma, where unconjugated antibodies (eg, rituximab, alemtuzumab, ofatumumab), radioimmunoconjugates (90Y-ibritumomab tiuxetan, 131I-tositumomab), and a drug conjugate (gemtuzumab ozogamicin) received approval from the US Food and Drug Administration (FDA). Another antibody-drug conjugate (ADC), brentuximab vedotin (SGN-35; anti-CD30-auristatin E), recently received accelerated FDA approval for Hodgkin lymphoma and anaplastic large cell lymphomas. There are also several other ADCs in preclinical and clinical development that target CD19, CD22, CD37, CD74, and CD79b.

[0311] Antibodies against all of these targets are logical choices for drug carriers, as they are internalized. The internalization and specificity of CD22 have made it a particularly important target for leukemia and lymphomas, with at least 3 different anti-CD22 conjugates in clinical investigation, including CMC-544 (acid-labile conjugated calicheamicin), an anti-CD22 conjugate. -CD22-maytansine (stably bound MCC-DM1) and CAT-3888 (formally BL22; a Pseudomonas exotoxin single-chain fusion protein). The active agent in all of these conjugates is subnanomolar in potency (ie, so-called ultratoxic).

[0312] Recently, we have developed methods for antibodies conjugated to SN-38, a topoisomerase I inhibitor with low nanomolar potency that is derived from the prodrug, irinotecan (Govindan et al, 2009, Clin Cancer Res. 15:6052- 62; Moon et al., 2008, J Med Chem 51:6916-26). Four SN-38 binding chemistries were initially examined using conjugates prepared with a slowly internalizing anti-CEACAM5 antibody (Govindan et al., 2009, Clin Cancer Res 15:6052-62; Moon et al., 2008, J Med Chem 51 : 6916-26). The conjugates retained CEACAM5 binding but differed in the rate of dissociation from SN-38 in human serum, with half-lives ranging from approximately 10 to 67 hours (Govindan et al., 2009, Clin Cancer Res 15:6052-62) . Finally, the linker designated CL2, with intermediate stability (~50% dissociated in 24-35 hours), was selected for further development. CL2 was recently modified by removing the phenylalanine in the cathepsin B cleavable dipeptide to simplify and improve manufacturing yields. The new derivative, designated CL2A, retains the pH-sensitive carbonate bond with SN-38, but is no longer selectively cleaved by cathepsin B. However, it has identical serum stability and in vivo activity as the CL2 linker. original (Cardillo et al., 2011, Clin Cancer Res 17: 3157-69). Since significant efficacy without toxicity was found with the slowly internalizing anti-CEACAM5-SN-38, we postulate that its activity was aided by the slow release of SN-38 from the antibody after it localized to a tumor. Therefore, the main objective of this report was to assess the therapeutic prospects of conjugates prepared using the CL2A linker with two antibodies that are highly specific for B-cell cancers but differ in their antigen expression and internalization properties.

[0313] Epratuzumab (Emab) is a rapidly internalizing (eg, >50% in 1 hour) humanized anti-CD22 IgG1 that has been extensively evaluated in lymphoma and leukemia in a conjugated or unconjugated form. Veltuzumab (Vmab) is a humanized anti-CD20 antibody that is also being studied clinically, but is slowly internalized (eg, ~10% in 1 hour). CD20 is generally expressed at much higher levels than CD22 in non-Hodgkin's lymphoma, whereas CD22 is preferentially expressed in acute lymphoblastic leukemia (ALL) but not in multiple myeloma. Both antibodies are effective in patients as unconjugated agents, but only veltuzumab is active in murine xenograft models (Stein et al., 2004, Clin Cancer Res 10:2868-76). Building on previous studies showing that 90 Y-Emab combined with unconjugated veltuzumab had higher efficacy in NHL models (Mattes et al., 2008, Clin Cancer Res 14:6154-60), we also examined Emab-SN -38 combination of Vmab, as this could provide additional benefit without competing for the same target antigen or having additional toxicity

Materials and methods

[0314] Cell lines. Ramos, Raji, Daudi (Burkitt's lymphomas), and JeKo-1 (mantle cell lymphoma) were purchased from the American Type Culture Collection. REH, RS4; 11, MN-60 and 697 (ALL) were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen. WSU-FSCCL (Follicular NHL) was a gift from Dr. Mitchell R. Smith (Fox Chase Cancer Center, Philadelphia, PA). All cell lines were grown in a humidified CO2 incubator (5%) at 37°C in recommended supplemented media containing 10 to 20% fetal calf serum and checked periodically for Mycoplasma.

[0315] Antibodies and conjugation methods. Epratuzumab and veltuzumab are humanized anti-CD22 and anti-CD20 IgG1 monoclonal antibodies, respectively. Labetuzumab (Lmab), a humanized anti-CEACAM5 IgG1, and RS7, a humanized anti-TROP-2 antibody (both from Immunomedics, Inc.), were used as irrelevant non-binding controls. Herein, Emab-SN-38, Vmab-SN-38, and Lmab-SN-38 refer to conjugates prepared using the CL2A linker as described above. In vitro studies in human serum showed that approximately 50% of the active SN-38 moiety is released from IgG each day (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Another linker, designated CL2E (see US Patent Nos. 7,999,083 and 8,080,250), is stable in human serum for 14 days, but contains a cathepsin B cleavage site to facilitate the release of SN-38 when processed in lysosomes. The method for preparing CL2E and the structures of the CL2A and CL2E linkers are given in US Patent Nos. 7,999,083 and 8,080,250. Conjugates contained approximately 6 SN-38 units per IgG (eg, 1.0 mg of IgG-SN-38 conjugate contains ~16 pg of SN-38).

[0316] Cell binding and cytotoxicity in vitro. Flow cytometry was performed using unconjugated specific and irrelevant antibodies incubated for 1 hour at 4°C, with binding revealed using fluorescein isothiocyanate (FITC)-Fc and goat-specific anti-human IgG (Jackson ImmunoResearch), also incubated for 1 hour at 4°C. Mean fluorescence was determined on a FACSCALIBUR® flow cytometer (Becton Dickinson) using a CellQuest software package.

[0317] Cytotoxicity was determined using the MTS dye reduction assay (Promega). Dose-response curves [COn/without Fc and goat anti-human F(ab')2; Jackson ImmunoResearch] is generated from the mean of triplicate determinations, and IC50 values were calculated using GraphPad PRISM® software (v5), with statistical comparisons using an F-test on best fit curves for the data. Significance was set at P<0.05.

[0318] Immunoblot. After 24 or 48 hours of exposure to test agents, markers of early (p21 expression) and late (PARP cleavage) apoptosis were revealed by Western blotting.

[0319] In vivo studies. The subcutaneous Ramos model was initiated by implantation of 1 x 10 7 cells (0.2 ml) of culture (>95% viability) into 4-6 week old female nude mice (Taconic). Three weeks after implantation, animals with tumors ranging from 0.4 to 0.8 cm3 (measured by caliper, L x W x D) were separated into groups of animals, each with the same range of tumor sizes. . Tumor size and body weights were measured at least weekly, and animals were withdrawn from the study when tumors grew to 3.0 cm3 or if they experienced a loss of body weight of 20% or more. The WSU-FSCCL and 697 intravenous models were initiated by intravenous injection of 2.5 x 106 and 1 x 107 cells, respectively, into female severe combined immunodeficient (SCID) mice (Taconic). Treatment began 5 days after WSU-FSCCL cell administration and 7 days after 697 inoculation. Animals were observed daily, using hindlimb paralysis or other signs of morbidity as surrogate survival endpoints. All treatments were administered intraperitoneally in <0.2 ml. Specific doses and frequency are given in the results section. Because mice convert irinotecan to SN-38 efficiently, the dose of irinotecan was adjusted based on SN-38 equivalents; SN-38 molar equivalents are based on 1.6 mass % ADC and 60 mass % irinotecan.

[0320] Efficacy was expressed on a Kaplan-Meier curve, using time to progression (TTP) as surrogate survival endpoints as above. Statistical analysis was performed by log-rank test using PRISM® GraphPad software (significance, P<0.05).

Results

[0321] Antigen expression and cytotoxicity in vitro. All cell lines were highly susceptible to SN-38, with EC50 values ranging from 0.13 nmol/L for Daudi to 2.28 nmol/L for RS4; 11 ( Table 10) . Except for 697 and RS4; 11, the anti-CD22 Emab-SN-38 conjugate was 2-7 times less effective than SN-38. This is a common finding with our targeted SN-38 conjugates as well as other non-targeted ones. Despite differences in antigen expression, Emab-CL2A-SN-38 and VmabCL2A-SN-38 had similar potencies to the non-binding anti-CEACAM5 conjugate Lmab-CL2A-SN-38, which was likely due to the approximately 90% dissociation of SN-38 during the 4-day MTS assay. Other in vitro procedures using shorter exposure times were also ineffective in discriminating differences in conjugate potencies. For example, annexin V staining after a 1-day exposure failed to find differences between treated and untreated cells (not shown).

Upregulation of p21 and PARP cleavage were also examined as early and late markers of apoptosis, respectively. Ramos did not express p21. However, PARP cleavage was detected, but only after a 48 hour exposure, and was more strongly expressed in SN-38 treated cells (not shown). The WSU-FSCCL cell line expressed p21, but neither upregulation of p21 nor cleavage of PARP was evident until 48 hours after exposure to Emab-CL2A-SN-38. However, both were observed after a 24 hour exposure with free SN-38 (not shown). While the enhanced intensity and earlier activation of apoptotic events with free SN-38 are consistent with its lower EC50 over the IgG-conjugated form, the results indicated that an exposure period of at least 48 hours would be required, but at this time, approximately 75% of the SN-38 would be released from the conjugate.

[0322] We again examined PARP cleavage and p21 expression, this time in cells treated with Emab-CL2A-SN-38 Vmab. Confirming the earlier study in Ramos, PARP cleavage first occurs only after a 48 hour exposure to the conjugate, with expression unchanged in the presence of a cross-linking antibody (not shown). Exposure to veltuzumab for more than 48 hours had no effect on PARP cleavage, but cleavage was strong at 24 hours when a cross-linking antibody (not shown) was added. However, when veltuzumab alone (without cross-linker) was combined with Emab-CL2A-SN-38, PARP cleavage occurred after a 24-hour exposure (not shown), indicating that veltuzumab might induce a shorter onset. rapid apoptosis, even in the absence of crosslinking. The only notable difference in the WSU-FSCCL cell line was that the combination greatly enhanced p21 expression at 48 hours (not shown), again suggesting an acceleration of apoptosis induction when veltuzumab is combined with the Emab-CL2A-SN-38 conjugate. The delay in apoptosis induction in WSU-FSCCL compared to Ramos is probably explained by the lower expression of CD22 and CD20.

[0323] Ultratoxic agents often use linkers that are highly stable in serum, as their premature release would increase toxicity, but these conjugates must be internalized for the drug to be optimally delivered. Because epratuzumab is rapidly internalized, we examined whether it might benefit from a more stably bound SN-38, by comparing the in vitro cytotoxicity of the CL2A-bound Emab-SN-38 conjugate with the stable CL2E-SN-38 conjugate in serum. . Both conjugates had similar binding affinity (not shown), but the more stable Emab-CL2E-SN-38 was approximately 40 to 55-fold less potent than the CL2A conjugate in 3 cell lines (not shown). While specificity was lacking with the CL2A conjugates, Emab-CL2ESN-38 was consistently approximately two-fold more potent than the non-binding Lmab-anti-CEACAM5-CL2E-SN-38 conjugate (not shown). We concluded that the more stably bound conjugate was unlikely to be appropriate for a slowly internalizing veltuzumab conjugate and therefore continued our investigation with only CL2A-linked SN-38 conjugates.

[0324] Due to the limitations of in vitro assays, efficacy was evaluated in xenograft models. As indicated in Table 10, all lymphoma cell lines have much higher expression of CD20 than CD22. Daudi had the highest expression of CD22 and CD20, but is highly sensitive in vivo to unconjugated veltuzumab, and in vitro testing revealed the highest sensitivity to SN-38 ( Table 10) . These properties would likely make it difficult to assess differences in activity attributed to conjugated SN-38 versus unconjugated antibody, particularly when unconjugated epratuzumab is not an effective treatment in animals. Because Ramos had previously been used to show an advantage in combining 90 Y-Emab with veltuzumab (Mattes et al., 2008, Clin Cancer Res 14:6154-60), we chose to start with a comparison of Emab-CL2ASN-38. and Vmab-CL2A-SN-38 conjugates in the Ramos human Burkitt cell line. Although flow cytometry showed a 15-fold higher expression of CD20 over CD22, immunohistology of Ramos xenografts showed abundant CD22 and CD20, with CD22 apparently more uniformly expressed than CD20 (not shown).

[0325] Ramos xenografts in untreated animals progressed rapidly, reaching the termination size of 3.0 cm3 from their initial size of 0.4 cm3 in 6 days (not shown) and, as previously reported, neither veltuzumab nor epratuzumab appreciably affected the progression of established Ramos disease xenografts (Sharkey et al., 2009, J Nucl Med 50:444-53). Consistent with previous findings using other SN-38 conjugates, none of the animals treated with a 4-week, twice weekly, 0.5 mg/dose treatment regimen had appreciable weight loss. Both conjugates were highly effective in controlling tumor growth, and 80% or more of the animals had no evidence of tumor at the end of the 4-week treatment ( FIG. 6) . The 0.25 mg dose of Vmab-CL2A-SN-38 was better at controlling growth during the first 4 weeks, but at 0.5 mg, similar early growth control was observed for both conjugates. Thus, despite a 15-fold higher expression of CD20 than CD22, EmabCL2A-SN-38 compared favorably with Vmab-CL2A-SN-38. Therefore, the remaining studies focused on Emab-S CL2AN-38 alone or in combination with unconjugated veltuzumab.

[0326] Emab-CL2A-SN-38 dose-response and specificity. A dose-response relationship was observed for the specific Emab-CL2A-SN-38 and irrelevant Lmab-CL2A-SN-38 conjugates, but Emab-CL2A-SN-38 had significantly better growth control at 2 of the 3 levels tested. , and with a strong tendency to favor the specific conjugate at the intermediate dose ( FIG. 7) . Again, 0.25 mg of Emab-CL2A-SN-38 eliminated most tumors; here, 7 of 10 animals were tumor-free at the end of the 12-week follow-up period, with no change in body weight. Animals receiving irinotecan alone (6.5 pg/dose; approximately the same SN-38 equivalents as 0.25 mg conjugate) had a median survival of 1.9 weeks, with 3 of 11 animals free of irinotecan at the end of the study. tumor, which was not significantly different from the median survival of 3.45 weeks for the irrelevant Lmab-CL2A-SN-38 conjugate (P=0.452; FIG. 7C) .

[0327] In the 697 disseminated leukemia model, the median survival of saline-treated animals was only 17 days from tumor inoculation. Animals receiving unconjugated epratuzumab plus irinotecan (same molar equivalents of SN-38 as 0.5 mg conjugate) had the same median survival, while animals receiving 0.5 mg Emab-CL2A-SN-38 twice weekly beginning 7 days after tumor inoculation survived up to 24.5 days, significantly longer than untreated animals (P < 0.0001) or for unconjugated epratuzumab administered with irinotecan (P = 0.016) . However, Emab-CL2A-SN-38 was not significantly better than the irrelevant conjugate (median survival = 22 days; P = 0.304), most likely reflecting the low expression of CD22 in this cell line.

[0328] EMAB-CL2A-SN-38 combined with unconjugated anti-CD20 Vmab. We previously reported better responses when 90Y-Emab was combined with unconjugated veltuzumab in the subcutaneous Ramos model (Mattes et al., 2008, Clin Cancer Res 14:6154-60) and thus this possibility was examined with Emab- CL2A-SN-38. In a pilot study, veltuzumab (0.1 mg), 0.1 mg Emab-CL2A-SN-38 was administered to 5 animals bearing subcutaneous Ramos tumors averaging approximately 0.3 cm3 were administered veltuzumab (0 0.1 mg), 0.1 mg Emab-CL2A-SN-38, or Emab-CL2A-SN-38 Vmab (all agents administered twice weekly for 4 weeks). The median TTP at 2.0 cm3 was 22, 14, and more than 77 days, respectively (veltuzumab vs Emab-CL2A-SN-38 alone, P = 0.59; Emab-CL2ASN-38 Vmab vs Emab- CL2A-SN-38, P = 0.0145), providing an early indication that the combination of veltuzumab with Emab-CL2A-SN-38 improved overall therapeutic response. In a follow-up study that also used a 4-week twice-weekly treatment regimen, 6 of 11 animals given 0.1 mg Emab-CL2A-SN-38 plus 0.1 mg veltuzumab did not showed evidence of tumors 16 weeks after onset. while median survival for animals receiving veltuzumab alone or with 0.1 mg of control Lmab-CL2A-SN-38 was 1.9 and 3.3 weeks, respectively, with 3 of 11 animals free of tumors at 16 weeks in each of these groups (not shown). Despite the longer median TTP and more survivors, no significant differences were found between groups. Therefore, in the Ramos model, which has abundant CD20 and moderate levels of CD22, the Emab-CL2A-SN-38 conjugate given at non-toxic dose levels was not significantly better than unconjugated anti-CD20 therapy, but the addition of Emab-CL2A-SN-38 to unconjugated anti-CD20 therapy appeared to improve response without toxicity. It is important to emphasize that sN-38 conjugates are administered at levels much lower than their maximum tolerated dose, and therefore these results should not be interpreted to mean that unconjugated anti-CD20 therapy is the same as conjugate. Emab-CL2A-SN-38.

[0329] Two additional studies were performed in an intravenously implanted model using the follicular NHL cell line WSU-FSCCL which has low expression of CD20 and CD22 (not shown). Median survival time for saline-treated animals was 40 to 42 days from tumor implantation. Irinotecan alone (not shown), administered at a dose containing the same SN-38 equivalents as 0.3 mg ADC, increased median survival (49 vs. 40 days, respectively; P = 0.042), but 14 of 15 animals succumbed to disease progression on day 49, the same day the last 4 of the 15 animals in the saline group (not shown) were eliminated. Despite its relatively low CD20 expression, veltuzumab alone (35 pg twice weekly x 4 weeks) was effective in this model. Median survival increased to 91 days in the first study, with 2 cures (day 161), and to 77 days in the second, but no survivors after 89 days (veltuzumab alone vs saline treatment, P<0.001 in both studies ). Unconjugated epratuzumab (0.3 mg/dose) combined with irinotecan and veltuzumab had the same median survival as veltuzumab alone, suggesting that neither epratuzumab nor irinotecan contributed to the net response.

[0330] As expected, due to the low expression of CD22 by WSU-FSCCL, EMAB-CL2A-SN-38 alone was not as efficient as in Ramos. At the 0.15 mg dose, no significant benefit was seen over the saline group, but at 0.3 mg, median survival increased to 63 days, providing a significant improvement compared to saline-treated animals. (P = 0.006). The second study, using 0.3 mg Emab-CL2A-SN-38, confirmed longer survival compared to the saline group (75 vs. 40 days; P<0.0001). The specificity of this response was not apparent in the first study, where the median survival of the irrelevant conjugate Lmab-CL2A-SN-38 and Emab-CL2A-SN-38 was not different at the 0.15 or 0 dose levels. 0.3 mg (42 vs. 49 days and 63 vs. 63 days for Emab-CL2A-SN-38 vs. anti-CEACAM5-CL2A-SN-38 conjugates at 2 dose levels, respectively). However, in the second study, the 0.3 mg dose of Emab-CL2A-SN-38 provided significantly better survival over the irrelevant conjugate (75 vs. 49 days; P<0.0001). Once again, the difficulty in showing specificity in this model is probably related to low expression of CD22.

[0331] The combination of the specific Emab-CL2A-SN-38 with veltuzumab substantially increases survival, with evidence of more robust responses than the control Lmab-CL2A-SN-38. For example, in the first study, animals treated with veltuzumab plus 0.15 or 0.3 mg of the control conjugate had a median survival of 98 and 91 days, respectively, which was similar to that of veltuzumab alone (91 days ; not shown). However, veltuzumab plus 0.15 mg of the specific Emab-CL2ASN-38 conjugate increased median survival to 140 days. Although this improvement was not significantly greater than that of veltuzumab alone (P = 0.257), when the dose of Emab-CL2A-SN-38 was increased to 0.3 mg with veltuzumab, 6 of 10 animals remained alive at the end of the study. , which provided a significant survival advantage over the control conjugate plus veltuzumab (P = 0.0002). In a second study, the median survival for veltuzumab alone was shorter than in the first (77 vs. 91 days), however, the median survival for the veltuzumab control conjugate was again 91 days, which now provided a significant survival advantage over veltuzumab alone. (P < 0.0001). The combination of the specific conjugate Emab-CL2A-SN-38 with veltuzumab extended the median survival to 126 days, which was significantly longer than the median survival of 75 and 77 days for Emab-CL2A-SN-38 and veltuzumab alone, respectively (P < 0.0001 for each). However, in this study, it did not meet the requirements for statistical improvement over combination with the control anti-CEACAM5-CL2A-SN-38 conjugate (P = 0.078).

Discussion

[0332] In the last 10 years, ADCs have made substantial progress in cancer therapy, however, there have also been some setbacks. The gains came largely when researchers chose to examine agents that were too toxic to be used alone, but when coupled to an antibody, these so-called ultratoxics produced substantially improved responses in preclinical tests. The recent approval of brentuximab vedotin, an auristatin conjugate, in Hodgkin lymphoma and the clinical success with the trastuzumab-DM1 anti-HER2-maytansine conjugate as a single agent in unconjugated trastuzumab-refractory breast cancer suggests that these ADCs that contain ultratoxic agents are becoming accepted treatment modalities. However, conjugates prepared with agents that are themselves potent in the picomolar range may have an increased risk of toxicity, as the recent decision to withdraw gemtuzumab ozogamicin, the anti-CD33 calicheamicin conjugate, from the market suggests (Ravandi, 2011, J Clin Oncol 29: 349-51). Thus, the success of an ADC may depend on the identification of the appropriate chemistries to bind the drug and the antibody, as well as the definition of a suitable target that is sufficiently expressed to allow adequate and selective delivery of the cytotoxic agent.

[0333] A CL2A linker was developed for the coupling of SN-38 to IgG that allows SN-38 to be slowly released from the conjugate in serum (approximately 50% per day). With this linker, a slowly internalizing antibody could be an effective treatment, perhaps because the tumor-localized conjugate releases a sufficient amount of drug locally, even without internalizing. The CL2A linker was also recently used with an antibody to TROP-2 that was reported to be rapidly internalized (Cardillo et al., 2011, Clin Cancer Res 17:3157-69.). Thus, it appears that the slow release mechanism is beneficial for internalizing and non-internalizing antibodies.

[0334] In this report, we have extended our evaluation of the CL2A linker by comparing SN-38 conjugates prepared with epratuzumab, a rapidly internalizing anti-CD22 IgG, and veltuzumab, a slowly internalizing anti-CD20 IgG, for the treatment of B cell neoplasms. Previous studies with the murine progenitor of epratuzumab had indicated that most of the antibody is internalized within 1 hour and 50% of CD22 is reexpressed on the cell surface within 5 hours (Shih et al., 1994, Int J Cancer 56: 538-45). This process of internalization and reexpression would allow intracellular delivery that could compensate for the lower surface expression of CD22. Because many B-cell malignancies express much more CD20 than CD22, a CD20-targeted conjugate could deliver more moles of drug by releasing its toxic payload after localization to the tumor.

[0335] In vitro cytotoxicity studies could not discriminate the potency of specific conjugates or even an irrelevant conjugate due to the release of SN-38 from the conjugate in the media. In fact, SN-38 was only somewhat more potent than the conjugates, which may reflect its accelerated ability to enter the cell and activate topoisomerase I. Because other studies revealed that the conjugates required 48-hour exposure before The first signs of apoptosis could be observed, we concluded that in vitro tests could not discriminate the potency of these 2 conjugates and, therefore, we resorted to in vivo studies.

[0336] In xenograft models, both conjugates had similar antitumor activity against Ramos tumors, which flow cytometry had indicated expressed nearly 15-fold more CD20 than CD22. This supported the selection of the anti-CD22-CL2ASN-38 Emab conjugate especially because it could be combined with unconjugated anti-CD20 Vmab therapy without concern that either agent would interfere with the binding of the other agent. Indeed, if an anti-CD20-CL2A-SN-38 conjugate were used, the total dose of IgG protein administered would likely be below a level typically needed for effective treatments with unconjugated anti-CD20 antibodies, since the limiting toxicity of the dose would be driven by SN-38 content. Adding more unlabeled anti-CD20 to an anti-CD20-CL2A-SN-38 conjugate would risk reducing conjugate absorption and potentially diminishing its efficacy. However, as we previously showed in combination studies using radiolabelled epratuzumab with unconjugated veltuzumab, benefits can be derived from both agents given at their maximally effective and safe doses. In vitro studies showed that veltuzumab, even in the absence of crosslinking which is used to enhance signaling, accelerated apoptotic events initiated by Emab-CL2A-SN-38. Therefore, as long as the Emab-CL2A-SN-38 conjugate is as effective as the anti-CD20 conjugate, selecting the Emab-CL2A-SN-38 conjugate is a logical choice because it allows for more effective combination therapy, even in tumors in which one or both antigens have low expression.

[0337] Because most ADCs using ultratoxic drugs are stably bound, we also tested a stable, but intracellularly cleavable serum anti-CD22-CL2E-SN-38 conjugate, but found it to be 40- to 55-fold less potent than with the CL2A linker. Others have examined a variety of ultratoxic drugs conjugated with anti-CD20 or anti-CD22 antibodies, finding that internalizing conjugates are generally more active, but have also noted that even slowly internalizing antibodies could be effective if the released drug penetrated the cell. cellular membrane. While the CL2A-type linker may be appropriate for SN-38, it may not be optimal for a more toxic agent, where even a small sustained release into serum would increase toxicity and compromise the therapeutic window.

[0338] EMAB-CL2A-SN-38 was active at a cumulative dose of 0.6 mg in mice bearing Ramos (75 pg two times per week for 4 weeks), which extrapolates to a human dose of only 2.5 mg/kg. Therefore, Emab-CL2A-SN-38 should have a wide therapeutic window in patients. In addition, an effective and safe dose of anti-TROP-2-CL2A-SN-38 conjugate was combined with a maximum tolerated dose of a 90 Y-labeled antibody without appreciable increase in toxicity but with improved efficacy (Sharkey et al ., 2011, Mol Cancer Ther 10: 1072-81). Therefore, the safety and efficacy profile of these SN-38 antibody conjugates are highly favorable for other combination therapies.

[0339] Although irinotecan is not routinely used for the treatment of hematopoietic cancers, SN-38 was as potent in lymphoma and leukemia cell lines as it was in solid tumors (Cardillo et al, 2011, Cancer Clin Res.

17: 3157-69.). In the WSUFSCCL cell line, the specific and irrelevant IgG conjugates were significantly better than irinotecan, while in Ramos, the median TTP with the irrelevant conjugate was longer but not significantly better than irinotecan. These results are consistent with other studies that have shown that nonspecific IgG is an excellent drug carrier and more potent in vivo than free drug or conjugates prepared with albumin or polyethylene glycol (PEG)-Fc. Although the PEG-SN-38 conjugate had significant antitumor effects, it was administered in its maximally tolerated amounts, ranging from 10 to 30 mg/kg SN-38 equivalents (Sapra et al., 2009, Haematologica 94: 1456-9) . In contrast, the maximum cumulative dose of SN-38 administered over 4 weeks to Ramos animals was only 1.6 mg/kg (i.e., the 0.25 mg dose of Emab-SN-38 administered twice per week for 4 weeks) and was not toxic.

[0340] The specific therapeutic activity of EMAB-CL2A-SN-38 appears to be enhanced in cell lines with higher expression of CD22. For example, in Ramos, specific therapeutic effects of Emab-CL2A-SN-38 were recorded only at 2 of 3 different dose levels examined, and a considerable number of tumors were completely ablated. In contrast, in WSU-FSCCL that had approximately 2.5-fold lower CD22 expression, Emab-CL2A-SN-38 significantly improved survival compared with the irrelevant anti-CEACAM5-CL2A-SN-38 conjugate in 1 of 2 studies. However, it is important to emphasize that when used in combination with unconjugated anti-CD20 therapy, Emab-CL2A-SN-38 amplifies the therapeutic response. Therefore, the combination of these two treatments could increase the response even in situations in which CD22 is not highly expressed.

[0341] In conclusion, using the less stable CL2A-SN-28 linker, the Emab anti-CD22-CL2A-SN-38 conjugate was equally active at non-toxic doses in vivo as a similar anti-CD20-CL2A-SN conjugate. -38, despite the fact that CD20 expression was more than one log-fold higher than that of CD22. Therapeutic responses benefited by the combination of Emab-CL2A-SN-38 with unconjugated Vmab anti-CD20 therapy, even when CD22 expression was low, suggesting that the combination therapy could improve responses in various cancer malignancies. B cells when both antigens are present. Current studies suggest that this combination is highly potent in various preclinical models of lymphoma and leukemia, but appears to have less host toxicity.

Example 14. Anti-CD74-CL2A-SN-38 conjugates for the treatment of human CD74+ cancers

[0342] CD74 is an attractive target for antibody-drug conjugates (ADC), because it internalizes and recycles after antibody binding. CD74 is primarily associated with hematologic cancers, but is also expressed in solid cancers. Therefore, the utility of ADCs prepared with the humanized anti-CD74 antibody, milatuzumab, for therapy of CD74-expressing solid tumors was examined. Milatuzumab-doxorubicin and two milatuzumab-SN-38 conjugates with cleavable linkers (CL2A and CL2E), which differ in their stability in serum and how they release SN-38 into the lysosome, were prepared. CD74 expression was determined by flow cytometry and immunohistology. In vitro and in vivo therapeutic cytotoxicity studies were performed on the human cancer cell lines A-375 (melanoma), HuH-7 and Hep-G2 (hepatoma), Capan-1 (pancreatic) and NCI-N87 (gastric) and Raji Burkitt's lymphoma. Milatuzumab-SN-38 ADCs were compared to sN-38 ADCs prepared with anti-TROP-2 and anti-CEACAM6 antibodies in xenografts expressing their target antigens.

[0343] Milatuzumab-doxorubicin was more effective in the lymphoma model, whereas in A-375 and Capan-1, only milatuzumab-CL2A-SN-38 showed therapeutic benefit. Despite a much lower surface expression of CD74 than TROP-2 or CEACAM6, milatuzumab-CL2A-SN-38 had similar efficacy on Capan-1 as anti-TROP-2-CL2A-SN-38, but on NCIN87 , anti-CEACAM6 and anti-TROP-2 conjugates were superior. Studies in 2 hepatoma cell lines at a single dose level showed a significant benefit over animals treated with saline, but not against an irrelevant IgG conjugate. CD74 is a suitable target for ADC in some solid tumor xenografts, with efficacy largely influenced by the uniformity of CD74 expression, and with SN-38 conjugates linked to CL2A providing the best therapeutic responses.

Introduction

[0344] CD74, designated the invariant or Ii chain, is a type II transmembrane glycoprotein that associates with HLADR and inhibits the binding of antigenic peptides to the class Ii antigen-presenting structure. It serves as a chaperone molecule, targeting invariant chain complexes to endosomes and lysosomes, an accessory molecule in B-cell maturation, using an NF-kB-mediated pathway, and in T-cell responses through interactions with CD44 (Naujokas et al., 1993, Cell 74: 257-68), and is a receptor for the proinflammatory cytokine, macrophage migration inhibitory factor (Leng et al., 2003, J Exp Med 197: 1467-76) , which is involved in activating the cell survival and proliferation pathways.

[0345] In normal human tissues, CD74 is expressed primarily on B cells, monocytes, macrophages, dendritic cells, Langerhans cells, activated T cell subsets, and thymic epithelium (not shown), and is expressed on more than 90% B cell tumors (Burton et al., 2004, Clin Cancer Res 10:6606-11; Stein et al., 2004, Blood 104:3705-11). Early studies had conflicting data on whether CD74 is present on the membrane, in part because antibodies against the invariant chain were specific for the cytoplasmic portion of the molecule, but also because there are relatively few copies on the surface and their half-life in the cell surface is very short. Approximately 80% of CD74 on the cell surface is associated with the MHC II HLA-DR antigen (Roche et al., 1993, PNAS USA 90: 8581-85). Using the murine anti-CD74 antibody, LL1, the Raji Burkitt lymphoma cell line is estimated to be 4.8 x 104 copies/cell, but due to rapid intracellular transit, ~8 x 106 antibody molecules are internalized and catabolized per day (Hansen et al., 1996, Biochem J 320: 293-300). Thus, internalization of CD74 is highly dynamic, with antibody moving rapidly from the surface and being discharged into the cell, followed by re-expression of CD74 on the surface. Fab' internalization occurs as rapidly as IgG binding, indicating that bivalent binding is not required. Subsequent studies with a CDR-grafted version of murine LL1, milatuzumab (hLL1), found that the antibody could alter B-cell expression proliferation, migration, and adhesion (Stein et al., 2004, Blood 104:3705-11; Qu et al., 2002, Proc Am Assoc Cancer Res 43:255; Frolich et al., 2012, Arthritis Res Ther 14:R54), but the exceptional internalizing properties of anti-CD74 antibody made it an effective carrier for intracellular administration of cancer therapeutics (eg, Griffiths et al., 2003, Clin Cancer Res 9:6567-71). Based on preclinical efficacy and toxicology results, phase I clinical trials with milatuzumab-doxorubicin in multiple myeloma (Kaufman et al., 2008, ASH Annual Meeting Abstracts, 112: 3697), as well as non-Hodgkin's lymphoma and leukemia chronic lymphocytic, have been initiated.

[0346] Interestingly, CD74 is also expressed in nonhematopoietic cancers, such as gastric, renal urinary bladder, non-small cell lung cancer, certain sarcomas, and glioblastoma (eg, Gold et al., 2010, Int J Clin Exp Pathol 4: 1-12) and thus may be a therapeutic target for solid tumors expressing this antigen. Since a milatuzumab-doxorubicin conjugate was highly active in hematologic cancer models, it was a logical choice for this evaluation. However, we recently developed methods for coupling the potent inhibitor of topoisomerase I, SN-38, to antibodies. SN-38 is the active form of irinotecan, whose pharmacology and metabolism are well known. These conjugates have nanomolar potency on solid tumor cell lines and were found to be active with antibodies that were not actively internalized. Previous studies indicated a preference for a linker (CL2A) that allowed SN-38 to dissociate from the conjugate in serum with a half-life of ~1 day, rather than other linkers that were more or less stable in serum. However, given milatuzumab's unique internalizing ability, a new linker (CL2E) was developed that is very stable in serum, but can release SN-38 when introduced into the lysosome.

[0347] Current research examines the prospects for the use of these three anti-CD74 milatuzumab conjugates, one with doxorubicin, and two SN-38 conjugates, for effective therapy primarily against solid tumors.

Materials and methods

[0348] Human tumor cell lines. Raji Burkitt lymphoma, A-375 (melanoma), Capan-1 (pancreatic adenocarcinoma), NCI-N87 (gastric carcinoma), Hep-G2 hepatoma, and MC/CAR myeloma cell lines were purchased from the American Tissue Culture Collection (Manassas , GOES). The HuH-7 hepatoma cell line was purchased from the Japan Health Science Research Resources Bank (Osaka, Japan). All cell lines were grown in a humidified CO2 incubator (5%) at 37°C in recommended medium containing 10% to 20% fetal calf serum and supplements. Cells were passaged <50 times and checked regularly for mycoplasmas.

[0349] Antibodies and conjugation methods. Milatuzumab (anti-CD74 MAb), epratuzumab (anti-CD22), veltuzumab (anti-CD20), labetuzumab (anti-CEACAM5), hMN15 (anti-CEACAM6), and hRS7 (anti-TROP-2) are humanized monoclonal IgG1 antibodies. CL2A and CL2E linkers and their SN-38 derivatives were prepared and conjugated with antibodies as described in the Examples above. Milatuzumab-doxorubicin conjugates were prepared as previously described (Griffiths et al., 2003, Clin Cancer Res 9:6567-71). All conjugates were prepared by disulfide reduction of IgG, followed by reaction with the corresponding maleimide derivatives of these linkers. Spectrophotometric analyzes estimated the drug:IgG molar substitution ratio to be 5-7 (1.0 mg of protein contains -16 pg of SN-38 or 25 pg of doxorubicin equivalent).

[0350] Cell binding and cytotoxicity in vitro. Assays to compare cellular binding of conjugated and unconjugated milatuzumab to antigen-positive cells and cytotoxicity tests used the MTS dye reduction method (Promega, Madison, WI).

[0351] Flow cytometry and immunohistology. Flow cytometry was performed in a manner that provided an assessment of only membrane-bound cytoplasmic or membrane antigen. Immunohistology was performed on formalin-fixed paraffin-embedded subcutaneous tumor xenograft sections, stained without antigen retrieval methods, using antibodies at 10 pg/ml that were revealed with an anti-human IgG conjugate.

[0352] In vivo studies. Female nude mice (4-8 weeks old) or female SCID mice (7 weeks old) were purchased from Taconic (Germantown, NY) and used after a 1-week quarantine. All agents, including saline controls, were administered intraperitoneally twice weekly for 4 weeks. Specific doses are given in Results. Toxicity was assessed by weekly weight measurements. For the Raji Burkitt lymphoma model, SCID mice were injected intravenously with 2.5 x 10 6 Raji cells in 0.1 ml medium. Five days later, the animals received a single intravenous injection (0.1 ml) of the conjugate or saline (n=10/group). Mice were observed daily for signs of distress and paralysis, and were sacrificed when hindlimb paralysis developed, >15% initial weight loss, or if they were moribund (survival surrogate endpoints).

[0353] Subcutaneous tumors were measured using a two-dimensional caliper, and tumor volume (TV) calculated as L x W/2, where L is the longest diameter and w is the shortest. Measurements were made at least once a week, and animals were terminated when tumors grew to 1.0 cm 3 (ie, surrogate survival endpoint). Melanoma cell line A-375 (6 x 10 6 cells in 0.2 ml) was implanted into nude mice and therapy started when tumors averaged 0.23 ± 0.06 cm 3 (n=8/group). Capan-1 was implanted subcutaneously into nude mice using a tumor suspension pool of serially passaged tumors (0.3 ml of a 15% w/v tumor suspension) combined with 8 x 106 tissue culture cells. Treatments were started when VT averaged 0.27 ± 0.05 cm3 (n=10/group). NCI-N87 gastric tumor xenografts were initiated by injecting 0.2 ml of a 1:1 (v/v) mixture of matrigel and 1 x 107 terminal culture cells subcutaneously. Therapy was started when VT averaged 0.249 ± 0.045 cm3 (n=7/group). The same procedure was followed to grow Hep-G2 and HuH-7 hepatoma xenografts in nude mice. Therapy was started when Hep-G2 averaged 0.364 ± 0.062 cm3 (n=5/group) and Huh-7 averaged 0.298 ± 0.055 cm3 (n=5/group).

[0354] Efficacy is expressed in Kaplan-Meier survival curves, using the surrogate endpoints mentioned above to determine median survival times. Analysis was performed by log-rank test (Mantel-Cox) using Prism GraphPad software (LaJolla, CA), with significance of P<0.05.

Results

[0355] CD74 expression in human tumor cell lines and xenografts. Six cell lines derived from 4 different solid tumor types were identified as positive for CD74 based primarily on analysis of permeabilized cells ( Table 11) , as the MFI of membrane-only CD74 in solid tumor cell lines was most often <2-fold higher than background MFI (except melanoma cell line A-375). Surface CD74 expression on Raji was >5-fold higher than solid tumor cell lines, but total CD74 on permeabilized Raji cells was similar to most solid tumor cell lines.

Table 11. CD74 expression by flow cytometry expressed as mean fluorescent intensity (MFI) of milatuzumab-positive gated cells.

Surface_____________ Surface and cytoplasmic MFI ratio MFI ratio hLL1: hLL1:

Cell line hLL1 (bkdg)a bkgd hLL1 (bkgd)b Bkgd Panc CAc Capan-1 22 (12) 1.8 248 (5) 49.6 Gastric Hs746T 17 (8) 2.1 144 (5) 28.8 NCI-N87 5 (4) 1.3 220 (6) 36.7 Melanoma A-375 16 (3) 5.3 185 (6) 30.8 Hepatoma Hep-G2 9 (6) 1.5 156 (5) 31.2

HuH-7 8 (5) 1.6 114 (4) 28.5 Raji lymphoma 59 (3) 19.6 143 (5) 28.6 ND, not performed

Background aMFI from cells incubated with GAH-FITC only.

[0356] Immunohistology showed that Raji's subcutaneous xenografts had largely uniform and intense staining, with marked cell surface marking (not shown). The Hep-G2 hepatoma cell line had the most uniform uptake of solid tumors, with moderately strong staining, but predominantly cytoplasmic staining (not shown), followed by the A-375 melanoma cell line which had somewhat less uniform staining with staining more intense, however, mainly cytoplasmic expression (not shown). The gastric pancreatic carcinoma cell lines Capan-1 (not shown) and NCI-N87 (not shown) had moderate (Capan-1) to intense (NCIN87) CD74 staining, but it was not evenly distributed. The HuH-7 hepatoma cell line (not shown) had the least uniform and weakest staining.

[0357] Immunoreactivity of conjugates. Kd values for unconjugated milatuzumab, milatuzumab-CL2A-SN-38, and milatuzumab-CL2E-SN-38 conjugates were not significantly different, averaging 0.77 nM, 0.59 nM and 0.80 nM, respectively. Kd values for unconjugated milatuzumab and conjugated doxorubicin measured in the MC/Ca R multiple myeloma cell line were 0.5 ± 0.02 nM and 0.8 ± 0.2 nM, respectively (Sapra et al, 2008, Cancer Clinic Res. 14: 1888-1896).

[0358] In vitro drug release and serum stability of conjugates. The release mechanisms of SN-38 from the mercaptoethanol-capped linkers CL2A and CL2E were determined in an environment partially mimicking lysosomal conditions, namely low pH (pH 5.0) and in the presence or absence of cathepsin B. CL2E-SN-38 substrate was inert at pH5 in the absence of the enzyme (not shown), but in the presence of cathepsin B, cleavage at the Phe-Lys site proceeded rapidly, with a half-life of 34 min (not shown). Formation of active SN-38 requires intramolecular cyclization of the carbamate bond at the tenth position of SN-38, which occurred more slowly, with a half-life of 10.7 h (not shown).

[0359] As expected, cathepsin B had no effect on the release of active SN-38 at the CL2A linker. However, CL2A has a cleavable benzyl carbonate bond, which releases active SN-38 at a similar rate as CL2E linker at pH5.0, with a half-life of ~10.2 hrs (not shown). The milatuzumab-doxorubicin conjugate, which has a pH-sensitive acylhydrazone bond, had a half-life of 7 to 8 h at pH5.0 (not shown).

[0360] While all of these linkers release drug at relatively similar rates under lysosomally relevant conditions, they have widely different stabilities in serum. Milatuzumab-CL2A-SN-38 released 50% free SN-38 in 21.55 ± 0.17 h (not shown), consistent with other CL2A-SN-38 conjugates. The CL2E-SN-38 conjugate, however, was highly inert, with a half-life extrapolated to ~2100 h. The milatuzumabdoxorubicin conjugate released 50% of the doxorubicin in 98 h, which was similar to 2 other antibody-doxorubicin conjugates (not shown).

[0361] Cytotoxicity. A major issue related to the evaluation of these conjugates was the relative potency of free doxorubicin and SN-38 in hematopoietic and solid tumor cell lines. Our group previously reported that SN-38 was active in several B-cell lymphoma and acute leukemia cell lines, with potencies ranging from 0.13 to 2.28 nM (Sharkey et al., 2011, Mol Cancer Ther 11:224 -3. 4). The potency of SN-38 in 4 of the solid tumor cell lines that were later used for in vivo therapy studies ranged from 2.0 to 6 nM (not shown). Doxorubicin had a mixed response, with a potency of 3-4 nM in the Raji lymphoma and A-375 melanoma cell lines, but was nearly 10-fold less potent against the Capan-1, NCI-N87, and Hep G2 cell lines. Other studies comparing the potency of SN-38 with doxorubicin found: LS174T colon cancer, 18 vs. 18 (nM potency of SN-38 vs. doxorubicin, respectively); breast cancer MDA-MB-231.2 vs. 2 nM; SK-OV-4 ovarian cancer, 18 vs. 90 nM; Calu-3 lung adenocarcinoma, 32 vs. 582 nM; Pancreatic cancer Capan-2.37 vs. 221 nM; and NCI-H466 small cell lung cancer, 0.1 vs. 2 nM. Thus, SN-38 was 5- to 20-fold more potent than doxorubicin in 4 of these 6 cell lines, with similar potency in LS174T and MDA-MB-231. Taken together, these data indicate that doxorubicin is less effective against solid tumors than SN-38, while SN-38 appears to be equally effective against solid and hematopoietic tumors.

[0362] As expected, the 3 conjugated forms were often some order of magnitude less potent than the free drug in vitro, as both drugs are expected to be readily transported into cells, while drug conjugates require antibody to transport drug binding within the cell (not shown). The CL2A-linked SN-38 conjugate is an exception, as more than 90% of the SN-38 is released from the conjugate into the medium during the 4-day assay period (Cardillo et al., 2011, Clin Cancer Res 17:3157 -69; Sharkey et al., 2011, Mol Cancer Ther 11:224-34). Thus, even if the conjugate were rapidly internalized, it would be difficult to discern differences between free drug and drug bound to CL2A.

[0363] The performance of stable CL2E-linked SN-38 was comparatively well in the Raji cell line, compared to free SN-38, but had substantially (7 to 16-fold) lower potency in all 4 solid tumor cell lines. , suggesting the relatively low surface expression of CD74 may be playing a role in minimizing drug transport in these solid tumors. The milatuzumab-doxorubicin conjugate had substantial differences in potency compared to free doxorubicin in all cell lines, which was similar in magnitude to CL2E-SN-38 conjugates with free SN-38 in solid tumor cell lines. .

[0364] In the 6 additional cell lines mentioned above, the milatuzumab-CL2A-SN-38 conjugate was 9 to 60-fold more potent than the milatuzumab-doxorubicin conjugate (not shown), but again, this result was influenced by largely due to the fact that the CL2A-linked conjugate releases most of its SN-38 into the medium during the 4-day incubation period, whereas the doxorubicin conjugate would release at most 50% of its drug during this incubation period. Same time. Milatuzumab bound to CL2E was not examined in these other cell lines.

[0365] In vivo therapy of human tumor xenografts. Previous in vivo studies with milatuzumab-doxorubicin or SN-38 conjugates prepared with various antibodies had indicated that they were effective at doses well below their maximum tolerated dose (Griffiths et al, 2003, Clin Cancer Res. 9: 6567-71; Sapra et al., 2005, Clin Cancer Res 11: 5257-64; Govindan et al., 2009, Clin Cancer Res 15: 6052-61; Cardillo et al., 2011, Clin Cancer Res 17: 3157-69; Sharkey et al. al., 2011, Mol Cancer Ther 11:224-34) and therefore in vivo testing focused on comparing amounts similar, but fixed, values of each conjugate at levels that were well tolerated.

[0366] Initial studies first examined doxorubicin and SN-38 conjugates in a disseminated Raji model of lymphoma in order to measure how the milatuzumab-doxorubicin conjugate compared to the 2 SN-38 conjugates (not shown). All specific conjugates were significantly better than the untargeted labetuzumab-SN-38 conjugate or saline-treated animals, which had a median survival of only 20 days (P < 0.0001). Although in vitro studies indicated an advantage of up to 8-fold for SN-38 conjugates in Raji, the best survival was seen with the milatuzumab-doxorubicin conjugates, where all animals received a single dose of 17.5 mg/day. kg (350 pg) and 7/10 animals receiving 2.0 mg/kg (40 pg) were alive at the end of the study (day 112) (eg, 17.5 mg/kg dose of milatuzumab-doxorubicin vs milatuzumab-CL2A-SN-38, P = 0.0012). Survival is significantly reduced for the more stable CL2E-SN-38 conjugates (P < 0.0001 and P = 0.0197, 17.5 and 2.0 mg/kg doses for CL2A vs. CL2E, respectively), although studies in vitro suggested that both conjugates would release active SN-38 at similar rates when internalized.

[0367] Five solid tumor cell lines, starting with the melanoma cell line A-375, were examined as having the best in vitro response to both doxorubicin and SN-38. A-375 xenografts grew rapidly, with saline-treated control animals having a median survival of only 10.5 days (not shown). A dose of 12.5 mg/kg (0.25 mg per animal) twice weekly of the milatuzumabCL2A-SN-38 conjugate prolonged survival to 28 days (P = 0.0006), which was significantly better than the conjugate. control epratuzumab-CL2A-SN-38 that had a median survival of 17.5 days (P = 0.0089), the latter not being significantly different from animals treated with saline (P = 0.1967). The milatuzumab-CL2A conjugate provided significantly longer survival than the milatuzumab-CL2E-SN-38 conjugate (P = 0.0014), which had the same median survival of 14 days as its control epratuzumab-CL2E-SN-38 conjugate. 38. Despite administering a 2-fold higher dose of milatuzumab-doxorubicin than SN-38 conjugates, median survival was no better than that of saline-treated animals (10.5 days).

[0368] As with the A-375 melanoma model, in Capan-1, only the CL2A-linked SN-38 conjugate was effective, with a median survival of 35 days, significantly different from untreated animals (P <0.036) (not shown), even at a lower dose (5 mg/kg; 100 pg per animal) (P < 0.02). Neither milatuzumab-CL2E nor epratuzumab-CL2A-SN-38 conjugates, or a 2-fold dose of the milatuzumab-doxorubicin conjugate, provided any survival advantage (P = 0.44 vs. saline). Of note, in the same study with animals receiving the same dose of the internalizing antiTROP-2 CL2A-SN-38 conjugate (hRS7-SN-38; IMMU-132), median survival was equal to milatuzumab-CL2A-SN -38 (not shown). The hRS7-CL2A-SN-38 conjugate had previously been identified as an ADC of interest for treating a variety of solid tumors (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). The surface binding MFI for hRS7 on Capan-1 was 237 (not shown), compared to 22 for milatuzumab (see Table 11) . Thus, despite having substantially lower surface antigen expression, the milatuzumab-CL2A-SN-38 conjugate performed as well as the hRS7-CL2A-SN-38 conjugate in this model.

[0369] With the milatuzumab-doxorubicin conjugate having inferior therapeutic results in 2 of the solid tumor xenografts, attention turned to comparing milatuzumab-SN-38 conjugates to SN-38 conjugates prepared with other humanized antibodies against TROP-2 (hRS7) or CEACAM6 (hMN-15), which are more highly expressed on the surface of many solid tumors (Blumenthal et al., 2007, BMC Cancer 7:2; Stein et al., 1993, Int J Cancer 55 : 938-46). Three additional xenograft models were examined.

[0370] In the gastric tumor model, NCI-N87, animals receiving 17.5 mg/kg/dose (350 pg) milatuzumab-CL2A-SN-38 provided some improvement in survival, but did not meet the statistical significance compared to animals treated with saline (31 vs. 14 days; P = 0.0760) or non-binding anti-CD20-CL2A-SN39 veltuzumab conjugate (21 days; P = 0.3128) ( not shown). However, hRS7- and hMN-15-CL2A conjugates significantly improved median survival at 66 and 63 days, respectively (P = 0.0001). The MFI for surface expressed TROP-2 and CEACAM6 were 795 and 1123, respectively, much higher than CD74 which was only 5 (see Table 11) . Immunohistology showed relatively intense cytoplasmic expression of CD74 in the xenograft of this cell line, but more importantly, it was scattered, appearing only in distinct pockets within the tumor (not shown). CEACAM6 and TROP-2 were more uniformly expressed than CD74 (not shown), with CEACAM6 being more intensely present both cytoplasmically and on the membrane, and TROP-2 is found primarily on the membrane. Thus, the improved survival with anti-CEACAM6 and anti-TROP-2 conjugates most likely reflects both higher antigen density and more uniform expression on NCI-N87.

[0371] In the hepatoma cell line Hep-G2 (not shown), immunohistology showed very uniform expression with moderate CD74 cytoplasmic staining, and flow cytometry indicated relatively low surface expression (MFI = 9). The MFI with hMN-15 was 175 and immunohistology showed fairly uniform membrane and cytoplasmic expression of CEACAM6, with isolated pockets of very intense membrane staining (not shown). A study in animals bearing Hep-G2 xenografts found that milatuzumab-CL2A-SN-38 extended survival to 45 days compared to 21 days in the saline-treated group (P = 0.0048), while hMN-15-CL2A-SN-38 conjugate improved survival at 35 days. There was a trend in favor of the milatuzumab conjugate over hMN-15-CL2A-SN-38, but it did not reach statistical significance (46 vs. 35 days; P = 0.0802). However, the conjugate Veltuzumab-CL2A-SN-38 binding provided a survival advantage similar to that of the milatuzumab conjugate. We previously noted that therapeutic results with non-binding conjugates might be similar to those of the CL2A-bound specific conjugate, particularly at higher protein doses, but the titration of the control and specific conjugates is generally selectively revealed. Therefore, none of the specific conjugates provided a selective therapeutic benefit at these doses in this cell line.

[0372] In another study using the hepatoma cell line Huh-7 (not shown), which had similar surface expression, but slightly decreased cytoplasmic levels as Hep-G2 (see Table 11) , met the conjugate hMN- 15-SN-38 providing a longer survival advantage (35 vs. 18 days), although not significantly different, than the milatuzumab-CL2A conjugate (P = 0.2944). Although the hMN-15 and milatuzumab conjugates were significantly better than saline-treated animals (P = 0.008 and 0.009, respectively), again, neither conjugate was significantly different from the nontargeting veltuzumab-SN-38 conjugate. this dose level (P = 0.4602 and 0.9033, respectively). Surface expression of CEACAM6 was relatively low in this cell line (MFI=81), and immunohistology showed both CD74 (not shown) and CEACAM6 (not shown) to be very weak and widely dispersed.

Discussion

[0373] The antibody-drug conjugate (ADC) approach to tumor-selective chemotherapy is an area of considerable current interest (eg, Govindan et al, 2012, Expert Opin Biol Ther. 12:873-90; Sapra et al, 2011, Expert Opin Biol Ther 20: 1131-49 Recent clinical successes (Pro et al., 2012, Expert Opin Biol Ther 12: 1415-21; LoRusso et al., 2011, Clin Cancer Res 17: 437 -47) They have occurred largely with the adoption of supertoxic drugs in place of the conventional chemotherapeutic agents that had previously been used.However, the selection of the target, the antibody and the linker of the drug are factors that influence the optimal performance of an ADC For example, in the case of trastuzumab-DM1, HER2 is abundant in tumors expressing this antigen, the antibody is internalized, and the antibody itself has antitumor activity, all of which could combine to improve therapeutic outcome. In contrast, CD74 is expressed at a m level. much lower on the surface of cells, but its unique internal surface reexpression and surface reexpression properties have enabled a milatuzumab anti-CD74 ADC to be effective in hematopoietic cancer xenograft models even with a moderately toxic drug, such as doxorubicin (Griffiths et al., 2003, Clin Cancer Res 9:6567-71; Sapra et al., 2005, Clin Cancer Res 11:5257-64). Although doxorubicin is used more frequently in hematopoietic cancers, while SN-38 and other camptothecins are administered to patients with solid tumors, we decided to evaluate the usefulness of doxorubicin and SN-38 conjugates of milatuzumab in solid tumors. The milatuzumab-doxorubicin conjugate was effective in xenograft models of several hematologic cancers, leading to its clinical trial (NCT01101594 and NCT01585688), while several SN-38 conjugates were effective in hematologic and solid tumor models, leading to clinical trials. led to 2 new conjugates in phase I clinical trials in colorectal and various epithelial cancers (NCT01270698 and NCT01631552).

[0374] In vitro, unconjugated doxorubicin and SN-38 had similar potency as doxorubicin against the Raji lymphoma cell line, but SN-38 was more potent in a number of different solid tumor cell lines. Interestingly, in vivo, the milatuzumab-doxorubicin conjugate provided the best response in Raji compared to the milatuzumab-SN-38 conjugates. However, in Capan-1 and A-375, milatuzumab-doxorubicin was less effective than the CL2A-linked milatuzumab SN-38 conjugate, even though in vitro tests had indicated that A-375 was equally sensitive to doxorubicin. free than SN-38. Two other cell lines, MDA-MB-231 breast cancer and LS174T colon cancer, also had similar potency with free doxorubicin as SN-38 in vitro, but since In vitro tests indicated that SN-38 was equally effective in solid and hematologic cancers, and Since SN-38 is 5- to 20-fold more potent than doxorubicin in most solid tumor cell lines tested, we decided to focus on the 2 conjugates of milatuzumab-SN-38 for the therapy of solid tumors. However, to better assess the utility of milatuzumab-SN-38 conjugates, we include a comparative evaluation of SN-38 ADCs prepared with antibodies against other antigens that are present on a variety of solid tumors.

[0375] We had previously investigated therapeutic responses with the hRS7-linked internalized SN-38 anti-TROP-2 conjugate CL2A in the Capan-1 cell line (Cardillo et al, 2011, Cancer Clin Res. 17: 3157-69), and therefore the efficacy of milatuzumab and hRS7 SN-38 conjugates was compared. In this study, both conjugates significantly improved survival compared to control antibodies, and the CL2A-linked SN-38 conjugates of each were superior to the CL2E-linked conjugates. Since flow cytometry had indicated that TROP-2 expression was higher than that of CD74 on Capan-1, this result suggested that the transport capabilities of CD74, which were known to be exceptional, were more efficient than TROP- 2. However, it is well known that antigen accessibility (i.e., cytoplasmic, physiologic, and “binding site” membrane barriers) and distribution among cells within a tumor are critical factors influencing all forms of targeted therapy, particularly those that depend on the proper intracellular delivery of a product to individual cells (Thurber et al., 2008, Adv Drug Del Rev 60: 1421-34). In situations where the antigen is not expressed uniformly on all cells within the tumor, having a targeted agent that slowly releases its payload after localizing to the tumor, such as CL2A-linked conjugates, would allow the drug to be diffuse to untargeted bystander cells, thus improving their range of effectiveness. In fact, a high expression of antigen could potentially impeding tumor penetration based on the binding site barrier effect, but the extracellular release mechanism could provide a mechanism for drug to diffuse within the tumor. This mechanism is also believed to aid the efficacy of other conjugates we have examined using internalizing-poor antibodies, such as the anti-CEACAM5 and anti-CEACAM6 used here. Milatuzumab-based conjugates rely more on the direct interaction of the antibody with the tumor cell, taking advantage of the rapid internalization and reexpression of CD74 that can compensate for its lower abundance on the cell surface. However, this advantage would be mitigated when CD74 is widely dispersed within the tumor, and without a mechanism to retain the conjugate within the tumor, the benefit of slow drug release from the conjugate would be lost. A previous review of human gastrointestinal tumors by our group suggests that they often have high level expression with good uniformity (Gold et al., 2010, Int J Clin Exp Pathol 4: 1-12).

[0376] During our initial evaluation of suitable linkers for SN-38, a number of different derivatives were examined, including a 'CL2E'-type linker that was designed to be docked at the 20-hydroxyl position of SN-38, similar to CL2A linker. However, that antibody conjugate lacked sufficient antitumor activity and was not searched for. Given the exceptional internalizing properties of milatuzumab, we decided to review the SN-38 linker chemistry, hypothesizing that rapid internalization of a CD74 conjugate would improve the drug loading of a more stable conjugate. We hypothesized that if the leaving group was phenolic, this might promote cyclization, and therefore the CL2E linker was designed to bind at the phenolic position 10 of SN-38.

[0377] At baseline, the CL2E-linked SN-38 conjugate had a similar promising IC50 as the CL2A conjugate in the Raji cell line, which was consistent with the view that if rapidly internalized, both conjugates would release the active form of SN-38 at about the same speed. However, as already mentioned, the in vitro activity of the CL2A conjugate is largely influenced by the release of SN-38 into the medium and does not necessarily reflect the uptake by the intact conjugate. When the CL2E-linked conjugate was found to be much less potent in solid tumor cell lines than the CL2A conjugate, this suggested that the lower surface expression of CD74 affected SN-38 internalization through milatuzumab binding. However, when in vivo studies in Raji showed that milatuzumab-CL2A-SN-38 was superior to the CL2E conjugate, some other factor that would affect the efficacy of CL2E had to be considered. One possible explanation is that the design of the linker in CL2E-SN-38 leaves the 20-position of the drug underivatized, making the lactone group susceptible to ring opening. In fact, studies with irinotecan have shown that the potency of SN-38 is decreased by several factors, with lactone ring opening to the carboxylate form possessing only 10% of the potency of the intact lactone form. In contrast, CL2A-linked SN-38 is derivatized at the 20-hydroxyl position, a process that stabilizes the lactone group in camptothecins under physiological conditions. Therefore, it is likely that the lactone ring of SN-38 is protected from cleavage in the CL2A, but not in the CL2E conjugate. Therefore, destabilization of the lactone ring might have contributed to decreased CL2E efficacy in vivo. Since the in vitro stability studies and serum stability analysis were performed under acidic conditions, we do not have a direct measure of the carboxylate form of SN-38 in any of these conjugates.

[0378] In conclusion, in vitro and in vivo results indicate that the milatuzumab-doxorubicin conjugate is superior to the CL2A-SN-38 conjugate in the Raji lymphoma cell line, which may reflect the improved stability of the doxorubicin conjugate compared to that of CL2A. However, the finding that the CL2A-SN-38 conjugate was more effective than the highly stable CL2E-SN-38 conjugate suggests that other issues, potentially related to drug activation or cell line sensitivities, may be at play. .

[0379] CD74 has multiple roles in cell biology; in antigen presenting cells, it may have a more dominant role in processing antigenic peptides, where solid tumors are found, its role could be more related to survival. These different roles could affect intracellular processing and trafficking. Alternatively, the lower efficacy of SN-38 bound to CL2E could reflect drug inactivation by lactone ring opening at SN-38, implying the importance of the specific linker. Finally, in solid tumor models, antigen accessibility appears to play a dominant role in defining the potency of milatuzumab-CL2A-SN-38 when measured against conjugates prepared with other internalizing (hRS7) or poorly internalizing (hMN15) antibodies. ) that were more accessible (surface expressed) and abundant. We suspect that this finding is universal for targeted therapies, but these studies have at least shown that the unique internalizing properties of a CD74-targeted agent can provide significant efficacy even when surface expression of the target antigen is minimal.

Example 15. Use of hRS7-SN-38 (IMMU-132) to treat therapy-refractive metastatic colon cancer (mCRC)

[0380] The patient was a 62-year-old woman with mCRC who originally presented with metastatic disease in January 2012. She underwent laparoscopic transverse ileal colectomy as first therapy a couple of weeks after diagnosis, and then received 4 cycles of FOLFOx chemotherapy ( leucovorin, 5-fluorouracil, oxaliplatin) in a neoadjuvant setting prior to right hepatectomy in March 2012 for removal of metastatic lesions in the right lobe of the liver. This was followed by an adjuvant FOLFOX regimen that was resumed in June 2012, for a total of 12 cycles of FOLFOX. In August, oxaliplatin was removed from the regimen due to worsening neurotoxicity. His last 5-FU cycle was on 09/25/12.

[0381] CT scan performed in January 2013 showed liver metastases. She was then evaluated as a good candidate for enrollment in the IMMU-132 (hRS7-CL2A-SN-38) research study. Comorbidities in her medical history include asthma, diabetes mellitus, hypertension, hypercholesterolemia, heart murmur, hiatal hernia, hypothyroidism, carpal tunnel syndrome, glaucoma, depression, restless legs syndrome, and neuropathy. His surgical history includes tube-ligation (1975), thyroidectomy (1983), cholecystectomy (2001), carpal tunnel release (2008), and glaucoma surgery.

[0382] At the time of entry into this trial, his target lesion was a 3.1-cm tumor in the left lobe of the liver. Non-target lesions included several hypoattenuated masses in the liver. His baseline CEA was 781 ng/m.

[0383] After the patient signed the informed consent, IMMU-132 was given on a schedule once a week by infusion for 2 consecutive weeks, then a break of one week, this constitutes a cycle of treatment. These cycles were repeated as tolerated. The first infusion of IMMU-132 (8 mg/kg) was started on February 15, 2013 and completed without notable events. She experienced nausea (grade 2) and fatigue (grade 2) during the course of the first cycle and has continued treatment since then with no major adverse events. He reported alopecia and constipation in March 2013. The first response evaluation performed (after 6 doses) on 04/08/2013 showed a reduction of the target lesion by 29% by computed tomography (CT). His CEA level decreased to 230 ng/mL on March 25, 2013. At the second response evaluation (after 10 doses) on May 23, 2013, the target lesion shrank by 39%, thus constituting a response. partial according to RECIST criteria. He has continued treatment since 06/14/13, receiving 6 cycles constituting 12 doses of hRS7-CL2A-SN-38 (IMMU-132) at 8 mg/kg. His general health and clinical symptoms have improved considerably since starting this investigational treatment.

Example 16. Use of hRS7-SN-38 (IMMU-132) to treat refractive therapy metastatic breast cancer

[0384] The patient was a 57-year-old woman with triple-negative stage IV (ER/PR negative, HER-neu negative) breast cancer originally diagnosed in 2005. She underwent a lumpectomy of the left breast in 2005, followed by dose-dense ACT in an adjuvant setting in September 2005. She then received radiation therapy, which was completed in November. Local disease recurrence was identified when the patient palpated a lump in the contralateral (right) breast in early 2012 and was then treated with CMF chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil). His disease recurred in the same year, with metastatic lesions in the skin of the chest wall. He then received a TAXOL® carboplatin chemotherapy regimen, during which thrombocytopenia resulted. His disease progressed and he was started on weekly doxorubicin, which was continued for 6 doses. The skin disease was also progressing. An FDG-PET scan on 09/26/12 showed disease progression in the chest wall and solid, enlarged axillary nodes. The patient received oxycodone for pain control.

[0385] He was given IXEMPRA® from October 2012 to February 2013 (every 2 weeks for 4 months), when the chest wall lesion broke open and bled out. He was then given XELODA®, which was not well tolerated due to neuropathy in his hands and feet, as well as constipation. The skin lesions were progressive and she was then enrolled in the IMMU-132 trial after giving her informed consent. The patient also had a medical history of hyperthyroidism and visual disturbances, with a high risk of CNS disease (however, brain MRI was negative for CNS disease). At the time of enrollment in this trial, her skin (target) lesions on the right breast measured 4.4 cm and 2.0 cm at largest diameter. She had another nontarget lesion in her right breast and an enlarged lymph node in her right and left axilla.

[0386] The first infusion of IMMU-132 (12 mg/kg) was started on March 12, 2013, which was well tolerated. Her second infusion was delayed due to a grade 3 (0.9) absolute neutrophil count (ANC) reduction on the scheduled infusion day one week later. After a one week delay and after receiving NEULASTA®, she was given her second IMMU-132, with a 25% dose reduction to 9 mg/kg. Since then, he has been receiving IMMU-132 as scheduled per protocol, once a week for 2 weeks, then a week off. Their first response evaluation on May 17, 2013, after 3 cycles of therapy, showed a 43% decrease in the sum of the long diameter of the target lesions, constituting a partial response by RECIST criteria. She is continuing treatment at the 9 mg/kg dose level. His general health and clinical symptoms have improved considerably since starting IMMU-132 treatment.

Example 17. Use of hRS7-SN-38 (IMMU-132) to Treat Refractory Metastatic Non-Small Cell Lung Cancer

[0387] This is a 60-year-old man diagnosed with non-small cell lung cancer. Patient receives chemotherapy regimens of carboplatin, bevacizumab for 6 months and shows a response, and then, after progressing, receives further cycles of chemotherapy with carboplatin, etoposide, TAXOTERE®, gemcitabine over the next 2 years, with occasional responses lasting no more than 2 months. The patient then presents with a 6.5 x 4 cm left mediastinal mass and pleural effusion.

[0388] After signing the informed consent, the patient receives IMMU-132 at a dose of 18 mg/kg every two weeks. Brief periods of neutropenia and diarrhoea, with 4 bowel movements in 4 hours, are experienced during the first two injections, but these resolve or respond to symptomatic medications within 2 days. After a total of 6 IMMU-132 infusions, CT evaluation of the index lesion shows a 22% reduction, just short of a partial response but definite tumor shrinkage. The patient continues with this therapy for another two months, when a partial response of 45% tumor shrinkage of the sum of the diameters of the index lesion is observed by CT, thus constituting a partial response according to the RECIST criteria.

Example 18. Use of hRS7-CL2A-SN-38 (IMMU-132) to Treat Refractory Metastatic Small Cell Lung Cancer

[0389] This is a 65-year-old woman with a diagnosis of small cell lung cancer, involving her left lung mediastinal lymph nodes, and MRI evidence of a left parietal cerebral lobe metastasis. Prior chemotherapy includes carboplatin, etoposide, and topotecan, but no response is seen. Radiation therapy also fails to control his disease. She is then given iMm U-132 at a dose of 18 mg/kg once every three weeks for a total of 5 infusions. After the second dose, he experiences hypotension and grade 2 neutropenia, which improves before the next infusion. After the fifth infusion, a CT study shows a 13% shrinkage of his target left lung mass. Brain MRI also shows a 10% reduction in this metastasis. She continues her IMMU-132 dose every 3 weeks for 3 months, and continues to show objective and subjective improvement in her condition, with a 25% reduction in left lung mass and a 21% reduction in brain metastasis.

Example 19. Therapy of a gastric cancer patient with metastatic stage IV disease with hRS7-CL2A-SN-38 (IMMU-132)

[0390] This patient is a 60-year-old man with a history of smoking and periods of heavy drinking over a 40-year period. She experiences weight loss, discomfort with eating and pain that is not relieved by antacids, frequent abdominal pain, low back pain, and more recently, palpable nodes in both armpits. He seeks medical advice and, after evaluation, is shown to have an adenocarcinoma, including some squamous features, at the gastroesophageal junction, based on biopsy through a gastroscope. Radiological studies (CT and FDG-PET) also reveal metastatic disease in the right and left axilla, mediastinal region, lumbar spine, and liver (2 tumors in the right lobe and 1 in the left, all between 2 and 4 cm in diameter) . Her gastric tumor is resected, and she is then given a course of chemotherapy with epirubicin, cisplatin, and 5-fluorouracil. After 4 months and a 6-week rest period, he is switched to docetaxel chemotherapy, which also fails to control his disease, based on progression confirmed by CT measurements of metastatic tumors and some general deterioration.

[0391] The patient then receives IMMU-132 (hRS7-CL2A-SN-38) therapy at a dose of 10 mg/kg infused every other week for a total of 6 doses, after which CT scans to assess your disease status. These infusions are well tolerated, with some mild nausea and diarrhea, accompanied by symptomatic medications. CT studies reveal that the sum of his index metastatic lesions has decreased by 28%, so he continues this therapy for another 5 courses. Follow-up CT studies show that the disease remains reduced by 35% by RECIST criteria from his baseline measurements prior to IMMU-132 therapy, and his general condition also appears to have improved, with the patient regaining an optimistic attitude toward control of your disease.

Example 20. Therapy of a patient with advanced colon cancer refractory to previous chemoimmunotherapy, using only IMm U-130 (labetuzumab-CL2A-SN-38).

[0392] The patient was a 50-year-old man with a history of stage IV metastatic colon cancer, first diagnosed in 2008 and undergoing colectomy and partial hepatectomy for primary and metastatic colon cancers, respectively. He then received chemotherapy, as indicated in FIG. 8, which included irinotecan, oxaliplatin, FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, oxaliplatin), and bevacizumab, as well as bevacizumab combined with 5-fluorouracil/leucovorin, for nearly 2 years. Thereafter, she was given cycles of cetuximab, either alone or in combination with FOLFIRI (leucovorin, 5-fluurouracil, irinotecan) chemotherapy for the next year or longer. In 2009, he received radiofrequency ablation therapy on his liver metastases while undergoing chemoimmunotherapy, and in late 2010 he underwent a wedge resection of his lung metastases, which was repeated a few months later in early 2011. Despite receiving chemoimmunotherapy in 2011, new lung metastases appeared at the end of 2011, and in 2012, lung and liver metastases were visualized. His baseline plasma carcinoembryonic antigen (CEA) titer was 12.5 ng/ml just before undergoing IMMU-130 antibody-drug therapy. The index lesions chosen by the radiologist to measure tumor size change by computed tomography were the right lung middle lobe and liver metastases, both totaling 91 mm as the sum of their longest diameters at baseline. before IMMU-130 (anti-CEACAM5-CL2A-SN-38).

[0393] This patient received 16 mg/kg doses of IMMU-130 by slow IV infusion every two weeks for a total of 17 treatment doses. The patient tolerated the therapy well, presenting only grade 1 nausea, diarrhea and fatigue. after the first treatment, which occurred after treatments 4 and 5, but not after, because he received medication for these side effects. After treatment 3, she did show alopecia (grade 2), which was present during subsequent therapy. Nausea, diarrhea, and occasional vomiting lasted only 2-3 days, and her fatigue after the first infusion lasted 2 weeks. Otherwise, the patient tolerated therapy well. Due to the long duration of receipt of this humanized (CDR-grafted) SN-38-conjugated antibody, anti-labetuzumab antibody was measured in her blood and none was detected, even after 16 doses.

[0394] The first computed tomography (CT) measurements were made after 4 treatments, and showed a 28.6% change from the sum of the measurements made at baseline, before this therapy, in the index lesions . After 8 treatments, this reduction became 40.6%, thus constituting a partial remission according to the RECIST criteria. This response was maintained for another 2 months, when their CT measurements indicated that the index lesions were 31.9% less than baseline measurements, but somewhat higher than the previously measured 40.6% decrease. Therefore, based on careful CT measurements of the index lesions in the lung and liver, this patient, who had failed prior chemotherapy and immunotherapy, including irinotecan (parent molecule of SN-38), showed an objective response to The active metabolite of irintotecan (or camptotechin), SN-38, when targeted through the humanized anti-CEACAM5 antibody, labetuzumab (hMN-14). It was surprising that although irinotecan (CPT-11) acts by releasing SN-38 in vivo, the SN-38-conjugated anti-CEACAM5 antibody was shown to be effective in a patient with colorectal cancer by inducing a partial response after the patient had previously failed to respond to his last therapy containing irinotecan. The reduction in the patient's plasma CEA titer also corroborated the CT findings: it fell from the initial level of 12.6 ng/ml to 2.1 ng/ml after the third dose of therapy, and was between 1.7 and 3.6 ng/mL between doses 8 and 12. Normal plasma CEA titer is generally considered to be between 2.5 and 5.0 ng/mL, so this therapy effected a normalization of his CEA titer in the blood.

Example 21. Therapy of a patient with advanced colon cancer with IMMU-130

[0395] This patient is a 75-year-old woman, initially diagnosed with metastatic colon cancer (stage IV). He undergoes a right partial hemicolectomy and resection of his small intestine and then receives FOLFOX, FOLFOX bevacizumab, FOLFIRI ramucirumab and FOLFIRI cetuximab therapies for a year and a half, when he shows progression of the disease, with dissemination of the disease to the posterior fornix , omentum, with ascites in the pelvis and a pleural effusion in the right side of the chest cavity. His baseline cEa titer just prior to this therapy is 15 ng/mL. IMMU-130 (anti-CEACAM5-CL2A-SN-38) 6 mg/kg is given twice a week for 2 consecutive weeks, and then a week off (3-week cycle), for more than 20 doses, which is very well tolerated, without any significant haematological or non-haematological toxicity. At 2 months of therapy, his plasma CEA titer is modestly reduced to 1.3 ng/mL, but at the 8-week evaluation he shows a 21% shrinkage of the index tumor lesions, increasing to a 27% shrinkage. % at 13 weeks. Surprisingly, the patient's ascites and pleural effusion decreased (the latter disappearing) at this time, thus markedly improving the patient's general condition. The patient continues her investigational therapy.

Example 22. Gastric Cancer Patient With Stage IV Metastatic Disease Treated With IMMU-130

[0396] The patient is a 52-year-old male who sought medical attention for gastric discomfort and pain related to eating for approximately 6 years and weight loss for the past 12 months. Palpation of the stomach area reveals a firm lump which is then gastroscopy revealing an ulcerous mass in the lower part of the stomach. A biopsy is performed and it is diagnosed as a gastric adenocarcinoma. Laboratory tests reveal no specific abnormal changes, except that liver function tests, LDH, and CEA are elevated, the latter being 10.2 ng/mL. Next, the patent undergoes a full-body PET, which reveals, in addition to the gastric tumor, metastatic disease in the left axilla and in the right lobe of the liver (2 small metastases). The patient's gastric tumor is resected, and then baseline CT measurements of his metastatic tumors are taken. Four weeks after surgery, he receives 3 cycles of combination chemotherapy consisting of a cisplatin and 5-fluorouracil (CF) regimen, but he does not tolerate it well, so he is switched to docetaxel. The disease appears to stabilize for about 4 months, based on the CT scans, but then the patient's complaints of increased weight loss, abdominal pain, loss of appetite, and extreme fatigue lead to repeated CT scans, which show an increase in the size of metastases. to the tune of 20% and a suspicious lesion at the site of the original gastric resection.

[0397] The patient then receives experimental therapy with IMMU-130 (anti-CEACAM5-CL2A-SN-38) on a weekly schedule of 8 mg/kg. She tolerates it well, but after 3 weeks she develops grade 2 neutropenia and grade 1 diarrhea. Her fourth infusion is postponed for a week, and then weekly infusions are reinstituted, with no evidence of diarrhea or neutropenia for the next 4 injections. The patient then undergoes a CT scan to measure the size of their metastatic tumor and view the original gastric resection area. The radiologist measured, according to the RECIST criteria, a decrease in the sum of metastatic lesions, compared to the baseline value prior to IMMU-130 therapy, of 23%. There does not appear to be any clear lesion in the area of the original gastric resection. The patient's CEA titer at this time is 7.2 ng/mL, which is greatly reduced from the pre-IMMU-130 baseline of 14.5 ng/mL. The patient continues weekly IMMU-130 therapy at the same dose of 8.0 mg/kg, and after a total of 13 infusions, his CT studies show that one liver metastasis has disappeared and the sum of all lesions metastatic is reduced by 41%. constituting a partial response of RECIST. The general condition of the patient improves and resumes normal activities while continuing to receive maintenance therapy of 8 mg/kg IMMU-130 every three weeks for another 4 injections. In the last blood CEA measurement, the value is 4.8 ng/mL, which is within the normal range for a smoker, which is the case in this patient.

Example 23. Therapy of relapsed triple negative metastatic breast cancer with hMN-15-CL2A-SN-38

[0398] A 58-year-old woman with metastatic triple-negative breast cancer previously treated with bevacizumab plus paclitaxel, without response, presenting with metastases to multiple ribs, lumbar vertebrae, solitary 3 cm diameter lesion in left lung, with considerable bone pain and fatigue. He is given experimental therapy with the humanized anti-CEACAM6 monoclonal antibody, hMN-15 IgG, conjugated to 6 molecules of SN-38 per IgG. He is given an infusion of 12 mg/kg every three weeks, repeated in 4 doses, as a course of therapy. Except for transient grade 2 neutropenia and some initial diarrhea, he tolerates therapy well, which is then repeated, after a 2-month break, for another course. The radiological examination indicates that he has a partial response according to the RECIST criteria, because the sum of the diameters of the index lesions decreases by 39%. His general condition, including bone pain, also improves and he returns to almost the same level of activity as before the illness.

Example 24. Therapy of relapsed, generally refractive, metastatic colon carcinoma with hMN-15-SN-38

[0399] A 46-year-old woman who has Stage IV metastatic colon cancer, with a prior history of resection of the main lesion who also had synchronous liver metastases to both lobes of the liver, as well as a single focus of spread to the right lung; These metastases measure, by CT, between 2 and 5 cm in diameter. He undergoes multiple courses of chemotherapy over a 3-year period, including 5-fluorouracil, leucovorin, irinotecan, oxaliplatin, cetuximab, and bevacizumab. On two occasions, there is evidence of disease stabilization or a short-term response, but no reduction of 30% or more in measured lesions. His plasma CEA titer at baseline before hMN-15-CL2A-SN-38 therapy is 46 ng/mL and his total index lesions measure a sum of 92 mm.

[0400] hMN-15-CL2A-SN-38 therapy is instituted at 12 mg/kg weekly for 2 weeks, with a one-week rest period thereafter, within a 21-day cycle. This cycle is repeated 3 times, with transient neutropenia and gastrointestinal side effects (nausea, vomiting, diarrhea). Surprisingly, despite not responding to FOLFIRI (which includes irinotecan or CPT-11) therapy, the patient shows a partial response by RECIST criteria after completing her therapy. He is then placed on a maintenance schedule of this therapy at a dose of 16 mg/kg once a month for the next 6 months. Subsequent scans show that her disease remains under control as a partial response (PR), and the patient is generally in good condition with a Kaaarnofsky performance status of 90%.

Example 25. Colon Cancer Patient With Stage IV Metastatic Disease Treated With Anti-CSAp-SN-38 Conjugate

[0401] This patient presents with colon cancer metastases to the left lobe of the liver and both lungs, after having a 9 cm sigmoid colon adenocarinoma resection, followed by chemoimmunotherapy with FOLIFIRI and cetuximab for 6 months, and then FOLFOX followed by bevacizumab for an additional period of approximately 9 months. Ten months after the initial resection and after the initiation of therapy, the stable disease believed to be present shows progression by the growth of lesions and the appearance of a new metastasis in the left adrenal gland. His plasma CEA at this time is 52 ng/mL and his general condition appears to have deteriorated, with abdominal pain, fatigue, and borderline anemia, suggesting possible internal bleeding.

[0402] You are now given a hmu-9 antibody CL2A-SN-38 conjugate (anti-CSAp) at a dose of 12 mg/kg per week for two weeks, with one week off, as a course of treatment, and then repeat for additional treatment cycles, measuring her blood count every week and receiving atropine medication for gastrointestinal reactions. Grade 2 alopecia is seen after the first cycle of treatment, but only grade 1 neutropenia. After 3 cycles of treatment, her plasma CEA titer is reduced to 19 ng/mL, and at this time her CT measurements show a decrease in index lesions in the liver and lungs by 24.1%. After 3 additional cycles of therapy, it shows a CT reduction of the index lesions of 31.4% and a decrease in size of the adrenal mass by approximately 40%. This patient is considered to be responsive to drug therapy with anti-CSAp-CL2A-SN-38 antibodies and is continuing on this therapy. His general condition appears to have improved, with less fatigue, no abdominal pain or discomfort, and generally more energy.

Example 26. Treatment of breast cancer with anti-CEACAM6-CL2A-SN-38 immunoconjugate

[0403] This patient has triple negative metastatic breast cancer (does not express estrogen receptors, progesterone receptor, or Her2/neu) that relapsed after several different therapies in the last 3 years. He presents several small tumors in both lungs, as well as metastases in the C4, C5, T2 and T3 vertebrae, as well as in several bilateral ribs. She is on standard therapy for her osteolytic lesions and is now starting treatment with hMN-15-CL2A-SN-38 at a dose of 16 mg/kg once a week for 3 weeks, with a break of one week, and then resume this treatment. 3-week cycle therapy twice more. At 2 weeks post therapy, CT scans are done to assess response and 2 of the small lung metastases are noted to have disappeared, while 1 of the larger lesions appears to have shrunk by approximately 40%. Metastases to the vertebrae are still present, but the C4 and C5 lesions appear smaller by approximately 25%. Of the rib metastases, 2 of 6 small lesions appear to be very small in size and it is unclear whether they are viable or small areas of scar or necrosis. The patient's tumor markers, as well as her LDH titers, appear to show stable or reduced levels, indicating that the progression of the disease has been arrested and there is also some evidence of disease reduction. Subjectively, the patient feels much better, with less fatigue and bone pain, and better breathing. She has experienced some nausea and vomiting after each therapy, which resolved within a week. The only other side effect has been transient thrombocytopenia, which also resolved within 7 days. She is under observation and will resume therapy cycles within 2 months.

Example 27. Treatment of metastatic colon cancer with anti-CEACAM5 and anti-CEACAM6-CL2ASN-38 combination immunoconjugates

[0404] This patient has metastatic colon cancer, with CT evidence of disease in the liver (5 cm lesion in the right lobe and 3 cm lesion in the left lobe), as well as 2 metastases (sizes 2 and 3 cm) to the right lung. The primary colon cancer was previously resected and the patient had cycles of postoperative therapy due to metachronous metastases to the liver and lungs. During therapy, liver metastases grow and one lung metastasis becomes two, making the patient a candidate for experimental chemoimmunotherapy. She is then started on a course of double antibody-drug conjugates, labetuzumab (hMN-14)-CL2A-SN-38 and hMN-15-CL2A-SN-38, each given every other day at a dose of 8 mg/kg. , once a week for 2 weeks and then repeated monthly for 4 months. Two weeks after therapy, the patient's condition is assessed by computed tomography and laboratory tests. CT scans reveal that the large tumor in the right hepatic lobe is reduced by 50%, the tumor in the left lobe by approximately 33%, and lung metastases by approximately 20% cumulatively for both tumors. His blood CEA titer decreased from 22 ng/ml at the start of treatment to 6 ng/ml at this follow-up. Subjectively, the patient states that she feels stronger and also appears to have more vigor in daily activities. Side effects are transient thrombocytopenia and leukopenia, which return to normal ranges within 2 weeks after therapy, and several episodes of nausea and vomiting, controlled with antiemetic medication. The patient is expected to resume these cycles of therapy in approximately 2 months, following another study to determine the status of the disease.

Example 28. Continuous infusion of antibody-drug conjugates

[0405] The patient was previously resected for rectal carcinoma and receives pre- and post-operative radiochemotherapy as per conventional treatment. He has been tumor free for four years, but now has 3 small metastatic lesions in the right lobe of the liver, discovered by routine CT and follow-up blood CEA values, increasing to 6.3 ng/mL from 3.0 ng/ml after initial treatment. An indwelling catheter and a continuous infusion of labetuzumab-CL2ASN-28 at a dose of 2 mg/kg for 17 days are placed. She then receives repeated continuous infusion therapy 5 weeks later, now for 3 weeks, at 1 mg/kg. Three weeks later, CT scans and CEA blood monitoring reveal that one of the liver metastases has disappeared and the other two are the same or slightly smaller. The CEA blood titer now measures 2.4 ng/ml. He has no symptoms and only experiences grade 2 nausea and vomiting during treatment, and grade 2 neutropenia, which resolves over time.

Example 29. Therapy of advanced metastatic colon cancer with anti-CEACAM5 immunoconjugate

[0406] The patient is a 50-year-old male who has not undergone prior therapies for metastatic colon cancer. The first line of therapy is FOLFIRINOX AVASTIN® (built in a staggered manner) starting with IROX (Irinotecan Oxaliplatin) in the first cycle. After starting this treatment, the patient has a CT scan that shows a decrease in the size of the liver metastases. This is followed by surgery to remove the tumor tissue. Adjuvant chemotherapy is a continuation of the first-line regimen (without the IROX part) that resulted in a transient period without recurrence. After an interval of about 1 year, a CT scan reveals recurrence of liver metastases. This leads to the start of the second-line regimen (FOLFIRI Cetuximab). Another CT shows a response in liver metastases. Radiofrequency ablation of liver metastases is then performed, followed by continuation of adjuvant chemotherapy with FOLFIRINOX Cetuximab, followed by maintenance Cetuximab for approximately one year. Another CT scan shows no evidence of disease. A new scan shows possible pulmonary nodules, which is confirmed. This leads to a wedge resection of the pulmonary nodules. Subsequently, FOLFIRI Cetuximab is restarted and continued. Subsequent CT scans show metastases in both the lung and liver.

[0407] At the time of administration of the hMN-14-CL2A-SN-38 immunoconjugate, the patient has advanced metastatic colon cancer, with metastases to both lungs and liver, that is insensitive to irinotecan (camptothecin). The hMN-14-CL2A-SN-38 immunoconjugate is administered at a dose of 12 mg/kg, repeated every two weeks. The patient shows a partial response with reduction of metastatic tumors by RECIST criteria.

[0408] Of note, only one patient in this 12 mg/kg (administered every other week) cohort shows hematologic grade 2 (neutropenia), and most patients have grade 1 or 2 nausea, vomiting, or alopecia, which are signs of activity of the antibody-drug conjugate, but well tolerated. The effect of the antibody moiety in enhancing camptothecin targeting explains the efficacy of the SN-38 moiety in cancer that had previously been resistant to unconjugated irinotecan.

Example 30. Treatment of metastatic pancreatic cancer with anti-MUC5AC-CL2A-SN-38 immunoconjugate

[0409] This 44-year-old patient has a history of metastatic pancreatic carcinoma, with inoperable pancreatic ductal adenocarcinoma in the head of the pancreas, and showing metastases to the left and right lobes of the liver, the former measuring 3 x 4 cm and the second measures 2 x 3 cm. The patient receives a cycle of gemcitabine but does not show an objective response. Four weeks later, hPAM4-CL2A-SN-38 is given iv at a dose of 8 mg/kg twice a week for 2 weeks, with a week off, and then repeated for another 2 cycles. CT scans are done one week later and show a total reduction in tumor mass (all sites) of 32% (partial response), along with a drop in her blood CA19-9 titer from 220 at baseline to 75 at the time of radiological evaluation. The patient exhibits only grade 1 nausea and vomiting after each treatment with the antibody-drug conjugate, and grade 2 neutropenia at the end of the last treatment cycle, which resolves 4 weeks later. No premedication is given to prevent infusion reactions.

Example 31. Use of hL243-CL2A-SN-38 to treat refractive therapy metastatic colon cancer (mCRC)

[0410] The patient is a 67-year-old man, presenting with metastatic colon cancer. Following transverse colectomy shortly after diagnosis, the patient receives 4 cycles of FOLFOX chemotherapy in a neoadjuvant setting prior to partial hepatectomy for removal of metastatic lesions in the left lobe of the liver. This is followed by an adjuvant FOLFOX regimen for a total of 10 FOLFOX cycles.

[0411] CT shows liver metastases. His target lesion is a 3.0 cm tumor in the left lobe of the liver. Non-target lesions included several hypoattenuated masses in the liver. Baseline CEA is 685 ng/mL.

[0412] After the patient signs the informed consent, hL243-CL2A-SN-38 (10 mg/kg) is administered every two weeks for 4 months. The patient experiences nausea (grade 2) and fatigue (grade 2) after the first treatment and continues treatment without major adverse effects. The first response evaluation performed (after 8 doses) shows a shrinkage of the target lesion by 26% by computed tomography (CT) and its CEA level decreases to 245 ng/mL. In the second evaluation of the response (after 12 doses), the target lesion has been reduced by 35%. His general health and clinical symptoms have improved considerably.

Example 32. Treatment of relapsed follicular lymphoma with IMMU-114-CL2A-SN-38 (anti-HLA-DR-SN-38)

[0413] After receiving R-CHOP chemotherapy for follicular lymphoma presenting with extensive disease in several regional lymph nodes (cervical, axillary, mediastinal, inguinal, abdominal) and spinal cord involvement, this 68-year-old man receives the experimental agent IMMU-114-CL2A-SN-38 (anti-HLA-DR-CL2A-SN-38) at a dose of 10 mg/kg weekly for 3 weeks, with a break of 3 weeks, and then a second course for another 3 weeks weeks. The change in the index tumor lesions is then evaluated by CT, showing a 23% reduction by CHESON criteria. The therapy is repeated for another 2 cycles, which then shows a 55% reduction of the tumor by CT, which is a partial response.

Example 33. Treatment of relapsed chronic lymphocytic leukemia with IMMU-114-CL2A-SN-38

[0414] A 67-year-old man with a history of CLL, as defined by the International Workshop on Chronic Lymphocytic Leukemia and World Health Organization classifications, presents with recurrent disease after prior therapies with fludarabine, dexamethasone, and rituximab , as well as a PVC scheme. He now has a fever and night sweats associated with generalized lymph node enlargement, reduced hemoglobin and platelet production, and a rapidly increasing white blood cell count. His l Dh is elevated and beta-2-microglobulin is nearly twice normal. The patient receives IMMU-114-CL2A-SN-38 conjugate therapy on a dosage schedule of 8 mg/kg weekly for 4 weeks, a 2-week break, and then the cycle is repeated again. Evaluation shows that the patient's hematology parameters are improving and his circulating CLL cells appear to be decreasing in number. Therapy is then resumed for another 3 cycles, after which her hematology and laboratory values indicate a partial response.

Example 34. Use of hMN-15-CL2A-SN-38 for the treatment of refractory, metastatic, non-small cell lung cancer

[0415] The patient is a 58-year-old man diagnosed with non-small cell lung cancer. He initially receives chemotherapy regimens of carboplatin, bevacizumab for 6 months and shows a response, and then, after progressing, he receives further cycles of chemotherapy with carboplatin, etoposide, TAXOTERE®, gemcitabine over the next 2 years, with occasional responses lasting no more than 2 months. The patient then presents with a 5.5 x 3.5 cm left mediastinal mass and pleural effusion.

[0416] After signing the informed consent, the patient receives hMN-15-CL2A-SN-38 at a dose of 12 mg/kg every two weeks. Brief periods of neutropenia and diarrhea are experienced during the first two injections, but these resolve or respond to symptomatic medications within 2 days. After a total of 6 hMN-15-SN-38 infusions, CT evaluation of the target lesion shows a 22% reduction. The patient continues this therapy for another two months, when CT shows a 45% partial response.

Example 35. Treatment of a patient with follicular lymphoma with hA19-CL2A-SN-38.

[0417] A 60-year-old man presents with abdominal pain and the presence of a palpable mass. The patient has CT and FDG-PET studies that confirm the presence of the mass with pathological adenopathies in the mediastinum, axillary lymph nodes, and neck. Laboratory tests are unremarkable except for elevated levels of LDH and beta-2-microglobulin. Bone marrow biopsy reveals several paratrabecular and perivascular lymphoid aggregates. These are lymphocytic with expression of CD20, CD19 and CD10 by immunostaining. The final diagnosis is grade 2 follicular lymphoma, stage IVA, with a FLIPI score of 4. The longest diameter of the largest affected node is 7 cm. The patient is administered a humanized anti-CD 19 IgG (hA19) monoclonal antibody conjugated to SN-38 (6 drug molecules per IgG) with a CL2A linker. The dose is 6 mg/kg weekly for 4 consecutive weeks, two weeks off, and then repeated cycles of 4 weeks of treatment every 6 weeks. After 5 cycles, bone marrow evaluations and CT scans show a partial response, where measurable lesions are decreased by approximately 60% and the bone marrow is much less infiltrated. In addition, LDH and beta-2-microglobulin titers also decrease.

Example 36. Treatment of relapsed precursor B cells with hA19-CL2A-SN-38

[0418] This 51-year-old woman has been undergoing therapy for precursor, Philadelphia chromosome negative, B-cell ALL, showing staining of the ALL cells for CD19, cD20, Cd10, CD38, and CD45. More than 20% of lymphoblasts in the bone marrow and blood express CD19 and CD20. The patient had received prior therapy with clofarabine and cytarabine, which resulted in considerable hematologic toxicity, but no response. A course of high-dose cytarabine (ara-C) was also started, but the patient was unable to tolerate it. He is given hA19-CL2A-SN-38 therapy in weekly doses by infusion of 6 mg/kg for 5 weeks, then 2 weeks off, with this therapy repeated two more times. Surprisingly, it shows an improvement in blood and marrow counts, sufficient to determine a partial response. After a 2-month break due to neutropenia (grade 3), therapy is resumed at 8 mg/kg every other week for another 4 cycles. At this time, she is much improved and is under consideration for maintenance therapy to try to get her to a stage where she may be a candidate for a stem cell transplant.

Example 37. Treatment of lymphoma with anti-CD22-CL2A-SN-38 immunoconjugate

[0419] The patient is a 62-year-old man with relapsed diffuse large B-cell lymphoma (DLBCL). After 6 cycles of RCHOP chemoimmunotherapy, he now has extensive nodal spread in the mediastinum, axillary and inguinal lymph nodes. She is given epratuzumab-CL2A-SN-38 (anti-CD22) at a dose of 12 mg/kg weekly x 3, with a week off, and then repeated again for another two cycles. One week later, the patient is evaluated by CT imaging and the total volume of the tumor is measured and shows a 35% decrease (partial response), which appears to be maintained for the next 3 months. Side effects are only thrombocytopenia and grade 1 nausea and vomiting after treatment, which resolves within 2 weeks. No prior therapy is given to reduce infusion reactions.

Example 38. Combination therapy of follicular lymphoma with veltuzumab and epratuzumab-CL2A-SN-38 in the first-line setting.

[0420] A 35-year-old woman was diagnosed with a low grade and good FLIPI scoring follicular lymphoma, presenting in her cervical lymph nodes, both axillae, and mediastinum. His spleen is not enlarged and the bone marrow biopsy does not reveal disease involvement. Symptomatically, she is not very affected, with only periods of high temperature, night sweats and somewhat more fatigued than usual. Her doctor decides not to do a watch-and-wait process, but to give this woman less aggressive therapy that combines a subcutaneous course of the humanized anti-CD20 monoclonal antibody, veltuzumab, weekly x 4 weeks (200 mg/m2) combined with two courses Weekly infusions of epratuzumab-CL2A-SN-38 anti-CD22, each infusion being a dose of 8 mg/kg. This combination therapy is repeated 2 months later, and after this, the patient is evaluated by CT and FDG-PET imaging studies, as well as a bone marrow biopsy. Surprisingly, a reduction of about 90% of all diseases is observed, and then another course of this combination therapy is given after a break of 4 weeks. Evaluation 4 weeks later shows a complete radiological response (and bone marrow biopsy). His doctor decides to repeat this course of therapy 8 months later, and radiological/pathological tests show a sustained complete remission.

Example 39. First line therapy of follicular lymphoma using veltuzumab-CL2A-SN-38

[0421] The patient is a 41-year-old woman who presents with low-grade follicular lymphoma, with measurable bilateral cervical and axillary lymph nodes (2-3 cm each), a 4-cm diameter mediastinal mass, and an enlarged spleen. She is given 3 cycles of veltuzumab-CL2A-SN-38 (anti-CD20-CL2A-SN-38) therapy, with each cycle consisting of a 10 mg/kg infusion once every 3 weeks. After completing therapy, CT measurements of her tumor show an 80% shrinkage. She is then given 2 additional cycles of therapy, and CT measurements indicate that a complete response is achieved. This is confirmed by FDG-PEt imaging.

Example 40. Therapy of relapsed DLBCL with humanized 1F5 antibody conjugated to CL2A-SN-38

[0422] A 53-year-old woman presents with recurrent diffuse large B-cell lymphoma at abdominal and mediastinal para-aortic sites 8 months after showing a partial response to R-CHOP chemotherapy administered for 6 cycles. She refuses further cytotoxic chemotherapy, so she is given milder therapy consisting of 10 mg/kg humanized anti-CD20 monoclonal antibody 1F5, conjugated to approximately 6 molecules of SN-28 per molecule of linker antibody. CL2A, once a week every two weeks for 5 infusions. CT and FDG-PET studies indicate a further reduction of his lymphomas by 40%, so after a 4-week rest period, therapy is resumed at a dose of 8 mg/kg every 3 weeks for a total of 5 infusions. Evaluation of his disease reveals a reduction of about 80%.

Example 41. Relapsed chronic lymphocytic leukemia therapy with rituximab-CL2A-SN-38

[0423] A 62-year-old man with an 8-year history of CLL, who has responded in the past to therapy with fludarabine, cyclophosphamide, and rituximab, and after a relapse to ibrutinib for a partial response lasting 9 months, was presents with progressive disease. The patient receives rituximab-CL2A-SN-38 monotherapy on a schedule of 12 mg/kg every 2 weeks for 3 cycles, reduced to 8 mg/kg every 2 weeks for another 4 cycles. A sustained improvement in cytopenias is observed, reflected by greater than 50% or a hemoglobin level greater than 11 g per deciliter, an absolute neutrophil count greater than 1500 cells per cmm, or a platelet count greater than 11k/cmm, which lasted about 9 months.

Example 42. First line therapy of DLBCL with veltuzumab-CL2A-SN-38 combined with bendamustine.

[0424] A 59-year-old man presents with multiple sites of DLBCL, including the chest, abdominal inguinal lymph nodes, and an enlarged spleen, as confirmed by CT, FDG-PET, and immunohistologic/pathologic diagnostics. Bendamustine is given at a dose of 90 mg/m2 on days 1 and 2, combined with veltuzumab-CL2A-SN-38 at a dose of 6 mg/kg on days 7 and 14, given every 4 weeks for four cycles. Radiological evaluation thereafter shows a partial response. After a 2-month break, therapy is repeated for another 2 cycles, and radiological evaluation shows a complete response. Cytopenias, primarily neutropenia, are manageable and do not reach a grade 3 level.

Example 43. First line therapy of mantle cell lymphoma (MCL) with veltuzumab-CL2A-SN-38 combined with lenalidomide.

[0425] The patient is a 68 year old male diagnosed with MCL after presenting with a GI complaint and lethargy. Colonoscopy reveals a 7-cm cecal mass, and examination reveals that he has stage IV disease. He is given combination therapy of lenalidomide, 25 mg orally daily on days 1 through 21 every 28 days. After two cycles, veltuzumabCL2A-SN-38 is given every other week at a dose of 10 mg/kg for 3 treatments, with a 2-week break. Then this is repeated again. Two weeks after completion of this therapy, the patient shows a partial response of his measured index lesion and reduction of other visualized lymph nodes. Four months later, lenalidomide therapy is repeated for 21 days, followed by 2 cycles of veltuzumab-SN-38. His illness is then shown to shrink further, though not a full response yet.

Example 44. Epratuzumab-SN-38 therapy of a patient with relapsed/refractory diffuse large B-cell lymphoma (DLBCL).

[0426] A 65-year-old man with symptoms of weight loss undergoes a biopsy of an epigastric mass, which is diagnosed as diffuse large B-cell lymphoma. He is treated with 6 cycles of standard R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). He has prolonged and persistent neutropenia (700 ANC) with mild thrombocytopenia (50-70k/mL), but no true anemia. Your IPI is high. The epigastric mass shows no change after this therapy, and he is put on a mild treatment regimen of anti-CD22 epratuzumab-CL2A-SN-38. This is a regimen of 4 mg/kg infused every two weeks for 4 infusions, then once every three weeks for another 3 infusions. His epigastric lymphoma is measured by CT one week later and shows a marked 52% reduction. The patient continues this treatment every three weeks for another 3 months, and continues to show this reduction in lymphoma mass with stabilization of his weight and improvement in his energy and activities.

Example 45. Humanized RFB4 therapy of a patient with relapsed follicular lymphoma

[0427] A 42-year-old woman presents with sharp, constant, severe pain in her lower abdomen that radiates to her back. Laboratory tests are normal, but an abdominal ultrasound shows a left lower anterior heterogeneous solid mass, measuring 7.5 x 6.2 x 7.0 cm. CT scans reveal a large mass within the mesentery of the left small intestine, with involvement of the adjacent lymph nodes. A CT-guided needle biopsy of the mass shows it to be a follicular lymphoma, grade 3. Immunohistochemistry shows a B-cell type with positive results for CD19, CD20, CD22, Bcl-2, and Bcl-6. PET studies reveal no disease above the diaphragm, in the bone marrow, or in the spleen, but bone marrow biopsy confirms lymphoma involvement. The patient undergoes 6 cycles of R-CHOP chemotherapy, resulting in a complete response to this therapy 4 months later. However, 10 months later, he relapses, with recurrence of the abdominal mass and adjacent lymph nodes, as well as an enlarged spleen and further bone marrow involvement as determined by PET scans and biopsy. She now begins a course of therapy with the humanized anti-CD22 monoclonal antibody, RFB4, conjugated to 6 SN-38 molecules by IgG using the CL2A linker, at a dose of 8 mg/kg weekly for 3 weeks, and then continued with 8 mg/kg. ./kg every two weeks for another 4 treatments. Two weeks later, he undergoes CT and FDG-PET studies, and his abdominal lesions and spleen show a 40% reduction and an overall decrease in bone marrow involvement. After a 4-week break, therapy is implemented at 4 mg/kg weekly for 4 weeks, followed by 6 mg/kg every other week for a further 5 treatments, with a further measurable reduction in the sum of the sizes of all cells. lesions measured by a total of 60%. The patient continues on hRFB4-CL2A-SN-38 maintenance therapy at 8 mg/kg once a month for the next 5 months and maintains her therapeutic response.

Example 46. Epratuzumab-CL2A-SN-38 therapy of a patient with relapsed/refractory acute lymphoblastic leukemia.

[0428] A 29-year-old male with CD22+ precursor B-cell acute lymphoblastic leukemia (ALL) has failed therapy with PEG-asparaginase, cyclophosphamide, daunorubicin, cytarabine (ara-C), vincristine, leucovorin, prednisone , methotrexate and 6-mercaptopurine, and supportive therapy with G-CSF (Neupogen), administered as induction/maintenance therapies according to a modified Larson protocol. The patient's leukemia is Philadelphia chromosome negative. Based on blood and bone marrow leukemia blast counts, the patient shows only a minimal response, with disease progression 4 months later. She is then given a weekly dose of epratuzumab-CL2A-SN-38 on an initial schedule of 6 mg/kg for 4 weeks, then reduced to 6 mg/kg every other week for 6 additional infusions. The patient is then evaluated by blood and marrow leukemic blasts, as well as by FDG studies of spleen size and bone marrow involvement, and appears to achieve a partial response, with concomitant improvement in signs and symptoms. patient general. This therapy is then continued for the next 8 months, but at 5 mg/kg every other week, with 3 weeks on and 2 weeks off, and a complete remission is achieved. The patient is now being evaluated as a candidate for a hematopoietic stem cell transplant.

Example 47. Humanized RFB4-CL2A-SN-38 therapy of a patient with relapsed/refractory acute lymphoblastic leukemia

[0429] After failing to respond to HIDAC (high-dose ara-C therapy), this 20-year-old man with precursor B-cell acute lymphoblastic leukemia is administered humanized anti-CD22 therapy with hRFB4 IgG conjugated to SN- 38 (average of 6 drug molecules per IgG), on a dosing schedule of 10 mg/kg per week for two weeks, then 1 week off, followed by infusions of 10 mg/kg every two weeks for 5 additional treatments. The patient is then evaluated for the presence of leukemic blast cells in the blood and bone marrow and shows >90% reduction. After a 4-week break, this course of therapy is repeated, and evaluation 4 weeks later shows a complete response with no minimal residual disease, as measured by PCR.

Example 48. Consolidation therapy with epratuzumab-CL2A-SN-38 in a DLBCL patient receiving R-CHOP chemotherapy

[0430] This 56-year-old woman with bilateral cervical adenopathy and cervical lymph nodes measuring 1.5 to 2.0 cm, as well as a 3 cm right axillary lymph node, as well as 2 cm bilateral retroperitoneal pelvic lymph nodes .5 to 3.0 cm, he is diagnosed with stage 3 diffuse large B-cell lymphoma, positive for CD20 and CD22. He is given a standard R-CHOP chemotherapy regimen given every 21 days with filgrastim and prophylactic antibiotics. After receiving 6 cycles of this therapy, the patient is given a 2-month rest period, and then placed on consolidation therapy with 8 mg/kg epratuzumab-CL2A-SN-38, infused every other week for 3 treatments. While the response after R-CHOP chemotherapy is minimal (less than 30% change in measured lesions), consolidation therapy with epratuzumab-CL2ASN-38 results in a partial response (>50% decrease in lesions). sum of all index injuries). After a 3-month break, this cycle of epratuzumab-CL2A-SN-38 therapy is repeated, re-administering the patient with filgrastim and prophylactic antibiotics, and her good remission is maintained.

Claims (15)

1. A method of producing a compound, CL2A- SN-38, of the structure,

which comprises performing a reaction scheme as shown: (i) PABOH, EEDQ. CHjCfc Lys(MMT)-PABOH _{Fmoc-Lys(MMT>OH}

8 (MCC-ino shown in box), copper(II) sulfate, sodium ascorbate,

CL2A-SN-38 wherein intermediate 9 is not exposed to triphenylphosphine. 2. The method of claim 1, further comprising using a 1.1-fold molar excess of tetrabutylammonium fluoride to remove a silyl protecting group and convert intermediate 6 to intermediate 7. 3. The method of claim 1, further comprising performing three washes of an organic extract comprising intermediate 6 with 0.05 M sodium acetate buffer, pH 5.3. 4. The method of claim 1, further comprising performing two washes of an organic extract comprising intermediate 7 with 0.25 M sodium citrate buffer, pH 6. 5. The method of claim 1, further comprising purifying intermediate 7 by stepwise elution from a silica gel column with a mixture of dichloromethane, ethyl acetate and methanol. 6. The method of claim 1, further comprising a) forming a biphasic mixture comprising, (i) copper sulfate and sodium ascorbate in water; and (ii) intermediate 7, intermediate 8 and 2,6-collidine in dichloromethane; Y b) stirring the biphasic mixture overnight at room temperature. 7. The method of claim 1, further comprising a) purify intermediate 9 by washing with EDTA and chromatography; Y b) concentrating intermediate 9 to give a solid for storage, before removal of a silyl group from intermediate 9 to form CL2A-SN-38. 8. The method of claim 7, further comprising (i) forming a solution of solid intermediate 9; and (ii) reacting intermediate 9 in solution with dichloroacetic acid and anisole to form CL2A-SN-38. 9. The method of claim 8, further comprising precipitating CL2A-SN-38 by dropwise addition of tert-butyl methyl ether (t-BME). 10. A method for the preparation of an SN38-conjugated protein or peptide comprising the method of claim 1 and further comprising reacting a maleimide moiety of CL2A-SN38 with a reduced sulfhydryl in a protein or peptide to produce a protein or peptide. peptide conjugated with SN38. 11. The method of claim 10, wherein the protein or peptide is an antigen-binding antibody or antibody fragment and the SN-38-conjugated antibody or antibody fragment is an immunoconjugate. 12. The method of claim 11, further comprising purifying the immunoconjugate by tangential flow filtration (TFF), wherein the TFF is performed with a 50,000 dalton molecular weight cut-off membrane using buffer diafiltration volumes of 25 to 30. 13. The method of claim 11, further comprising formulating the immunoconjugate in Good's biological buffer at a pH of 6.0 to 7.0 and lyophilizing the immunoconjugate for storage, wherein Good's biological buffer is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and 1,4 -piperazinediethanesulfonic acid (PIPES), in the pH range of 6-7, preferably in the pH range of 6.5 to 7, and at a buffer concentration of 10-100 mM. 14. The method of claim 11, wherein the antibody or antibody fragment binds between 6 and 8 copies of CL2ASN38. 15. The method of claim 11, wherein the antibody is selected from the group consisting of LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (anti-CD22), RS7 (anti-EGP-1), PAM4 ( anti-MUC5AC), KC4 (anti-mucin), A19 (anti-CD19), A20 (anti-CD20), MN-14 (anti-CEACAM5), MN-15 (anti-CEACAM6), MN-3 (anti- CEACAM6), R1 (anti-IGF-1 R), Mu-9 (anti-CSAp), Immu 31 (anti-AFP), CC49 (anti-TAG-72), J591 (anti-PSMA), HuJ591 (anti-PSMA) , AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX), and hL243 (anti-HLA-DR).