WO2018183494A1

WO2018183494A1 - Cd19-targeting antibody-drug conjugates

Info

Publication number: WO2018183494A1
Application number: PCT/US2018/024840
Authority: WO
Inventors: Stuart William HICKS; Jutta Deckert; Kathleen R. Whiteman
Original assignee: Immunogen Inc
Current assignee: Immunogen Inc
Priority date: 2017-03-31
Filing date: 2018-03-28
Publication date: 2018-10-04
Anticipated expiration: 2019-09-30
Also published as: WO2018183494A8

Abstract

The present invention is directed to anti-CD 19 antibody-drug conjugates and their use for the treatment of B-cell malignancies.

Description

CD19-TARGETING ANTIBODY-DRUG CONJUGATES

RELATED APPLICATION

This application claims the benefit of the filing date, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 62/480,217, filed on March 31, 2017, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to anti-CD 19 antibody-drug conjugates and their for the treatment of B-cell malignancies.

BACKGROUND OF THE INVENTION

Cell surface molecules expressed by B cells and their malignant counterparts represent important targets for immunotherapy. CD 19 is a cell surface membrane protein expressed in most mature and immature B-cell neoplasms, making it a promising target for antibody drug conjugate (ADC) therapy for B-cell malignancies.

B-cell non-Hodgkin's lymphoma (B-NHL) is the fifth most common malignancy in the United States and continues to increase in incidence, especially in elderly patients. While patients with hematological malignancies have benefited over the past decade from therapeutic optimization using conventional drug therapy, a majority of patients still succumb to their disease and drug therapies remain highly toxic.

Thus, there is a need for new therapies for B-cell malignancies with improved safety and efficacy. SUMMARY OF THE INVENTION

The present invention is based on the preclinical activity of a CD19-targeting ADC, huB4-DGN462 and huB4-DGN549. HuB4-DGN462 is an antibody-drug conjugate composed of a humanized IgGl monoclonal antibody, huB4, which specifically targets the CD 19 antigen, conjugated through a disulfide linker, sulfo-SPDB, to the cytotoxic benzodiazepine dimer compound DGN462, a DNA alkylating agent. huB4-DGN549 is an antibody-drug conjugate composed huB4 conjugated to the cytotoxic benzodiazepine dimer compound DGN549, a DNA alkylating agent In one embodiment, the present invention provides an anti-CD 19 antibody-drug conjugate represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein r is an integer from 1 to 10, Y is - S0₃H, and the antibody is an anti-CD 19 antibody that specifically binds to a CD 19 antigen and comprises:

i) a light chain CDR1 comprising SASSGVNYMH (SEQ ID NO: 1); a light chain CDR2 comprising DTSKLAS (SEQ ID NO: 2); and a light chain CDR3 comprising HQRGSYT (SEQ ID NO: 3); and

ii) a heavy chain CDR1 comprising SNWMH (SEQ ID NO: 4); a heavy chain CDR2 comprising EIDPSDSYTN (SEQ ID NO: 5); and a heavy chain CDR3 comprising

GSNPYYYAMDY (SEQ ID NO: 6).

In another embodiment, the present invention provides an anti-CD 19 antibody-drug conjugate represented by the following formula:

i) a light chain CDR1 comprising SASSGVNYMH (SEQ ID NO: 1); a light chain

CDR2 comprising DTSKLAS (SEQ ID NO: 2); and a light chain CDR3 comprising HQRGSYT (SEQ ID NO: 3); and

GSNPYYYAMDY (SEQ ID NO: 6).

In yet another embodiment, the present invention provides an anti-CD 19 antibody- drug conjugate represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein r_c is 1 or 2, Y is -SO₃H or a pharmaceutically acceptable salt thereof, and the antibody is an anti-CD 19 antibody that specifically binds to a CD 19 antigen and comprises:

i) a light chain CDR1 comprising SASSGVNYMH (SEQ ID NO: 1); a light chain CDR2 comprising DTSKLAS (SEQ ID NO: 2); and a light chain CDR3 comprising

HQRGSYT (SEQ ID NO: 3); and

GSNPYYYAMDY (SEQ ID NO: 6).

Also provided is a pharmaceutical composition comprising the conjugate of the present invention described herein and a pharmaceutically acceptable carrier.

The present invention also provides a method of treating a cancer in a mammal in need thereof comprising administering to the mammal a therapeutically effective amount of the conjugate of the present invention described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the anti-proliferative activity of huB4-DGN462 and its unconjugated toxin, DGN462SMe, in non-Hodgkin lymphoma cell lines. Cells were treated for 72h with a large range of concentration of a single agent. ALCL, anaplastic large cell lines (n=5); CLL, chronic lymphocytic leukemia (n=2); ABC-DLBCL, activated B cell like diffuse large B cell (n=7); GCB-DLBCL, germinal center B cell like diffuse large B cell (n=20); MCL, mantle cell lymphoma (n=10); MZL, marginal zone lymphoma (n=3); PMBCL, Primary Mediastinal Large B-Cell Lymphoma (n=l); SMZL, splenic marginal zone lymphoma (n=3) and others (n=6).

FIG. 2 shows in vivo efficacy of huB4-DGN462 in mice bearing DoHH2 tumor cells. FIG. 3 shows in vivo efficacy of huB4-DGN462 in mice bearing Farage tumor cells.

FIG. 4 shows in vitro cytotoxicity of huB4-DGN462 as compared to huB4-SPDB- DM4 in B-NHL and B-ALL cell lines.

FIG. 5 shows in vivo efficacy of huB4-s-SPDB-DGN462 and huB4-DGN549 in mice bearing DoHH2 tumor cells as compared huB4-SPDB-DM4.

FIG. 6 shows in vivo efficacy of huB4-s-SPDB-DGN462 and huB4-DGN549 in mice bearing OCI-Lyl8 tumor cells as compared huB4-SPDB-DM4.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

It should be understood that any of the embodiments described herein, including those described under different aspects of the invention (e.g., compounds, conjugates,

compositions, methods of making and using) and different parts of the specification

(including embodiments described only in the Examples) can be combined with one or more other embodiments of the invention, unless explicitly disclaimed or improper. Combination of embodiments are not limited to those specific combinations claimed via the multiple dependent claims.

Definitions

The term "compound" or "cytotoxic compound," "cytotoxic dimer" and "cytotoxic dimer compound" are used interchangeably. They are intended to include compounds for which a structure or formula or any derivative thereof has been disclosed in the present invention or a structure or formula or any derivative thereof that has been incorporated by reference. The term also includes, stereoisomers, geometric isomers, tautomers, solvates, metabolites, and salts (e.g., pharmaceutically acceptable salts) of a compound of all the formulae disclosed in the present invention. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of "stereoisomers," "geometric isomers," "tautomers," "solvates," "metabolites," "salt" "conjugates," "conjugates salt," "solvate," "hydrate," or "polymorph" in certain aspects of the invention described in this application shall not be interpreted as an intended omission of these forms in other aspects of the invention where the term "compound" is used without recitation of these other forms.

The term "chiral" refers to molecules which have the property of non- superimposability of the mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner.

The term "stereoisomer" refers to compounds which have identical chemical constitution and connectivity, but different orientations of their atoms in space that cannot be interconverted by rotation about single bonds.

"Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as crystallization, electrophoresis and chromatography.

"Enantiomers" refer to two stereoisomers of a compound which are non- superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and I or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

The term "tautomer" or "tautomeric form" refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include

interconversions by reorganization of some of the bonding electrons.

The term "conjugate" as used herein refers to a compound described herein or a derivative thereof that is linked to an anti-CD 19 antibody described herein.

The phrase "pharmaceutically acceptable salt" as used herein, refers to

pharmaceutically acceptable organic or inorganic salts of a compound of the invention.

Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate," ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e. , l,l '-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g. , sodium and potassium) salts, alkaline earth metal (e.g. , magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure.

Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.

The terms "cancer" refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells, and/or benign or pre-cancerous cells. Examples of cancers include, but are not limited to, B-cell malignancies or T-cell malignancies. In certain embodiments, the cancer is a leukemia or lymphoma. More particular examples of such cancers include the group consisting of B-cell lymphomas including non-Hodgkin' s lymphoma (NHL), precursor B-cell lymphoblastic leukemia/lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal type, and splenic marginal zone lymphoma (SMZL), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), activated B cell like diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B cell like diffuse B-cell lymphoma (GCB-DLBCL), Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder,

Waldenstrom's macroglobulinemia, anaplastic large-cell lymphoma (ALCL), primary mediastinal large B-cell lymphoma (PMBCL), and B-cell acute lymphoblastic leukemia (B- ALL). In certain embodiments, the cancer is B-ALL. In certain embodiments, the cancer is NHL, DLBCL, FL or MCL. In certain embodiments, the cancer is ABC-DLBCL or GCB- DLBCL.

Antibody-Drug Conjugates

The antibody-drug conjugates (ADCs) of the present invention comprises an anti-

CD 19 antibody linked to one or more cytotoxic compounds of the present invention via a disulfide linker, specifically the N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo- SPDB) linker:

In one embodiment, the cytotoxic compound of the present invention is represented by the following formu

or a pharmaceutically acceptable salt thereof, wherein Y is -SO₃H. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt or a potassium salt. In certain embodiments, Y is -S0₃Na or -S0₃K. In certain embodiments, Y is -S0₃Na.

In certain embodiments, the antibody-drug conjugate of the present invention is:

or a pharmaceutically acceptable salt thereof, wherein the antibody is an anti-CD 19 antibody, r is an integer from 1 to 10, and Y is -S0₃H. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt or a potassium salt. In certain embodiments, Y is -S0₃Na or -S0₃K. In certain embodiments, Y is -S0₃Na.

In certain embodiments, DGN462 is the compound of formula (sDl). In certain embodiments, DGN462 is the compound of formula (Dl). In certain embodiments, DGN462 is a mixture of compound Dl and compound sDl .

In some embodiments, the antibody-drug conjugates (ADCs) of the present invention comprises an anti-CD 19 antibody linked directly to one or more cytotoxic compounds of the present invention. In one embodiment, the cytotoxic compound of the present invention is represented by the follo ing formula:

In certain embodiment, DGN549 is compound of formula sD2. In certain

embodiments, DGN549 is the compound of formula (D2). In certain embodiments, DGN549 is a mixture of compound D2 and compound sD2.

or a pharmaceutically acceptable salt thereof, wherein the antibody is an anti-CD 19 antibody, r is an integer from 1 to 10, and Y is -SO₃H. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt or a potassium salt. In certain embodiments, Y is -S0₃Na or -S0₃K. In certain embodiments, Y is -S0₃Na. The cytotoxic compounds and the antibody-drug conjugates shown above can be prepared using the methods and procedures disclosed in International Patent Application Publication No. WO 2016/036801, which is incorporated herein by reference in its entirety.

In certain embodiments, the conjugates described herein may comprise 1-10 cytotoxic compounds, 2-9 cytotoxic compounds, 3-8 cytotoxic compounds, 4-7 cytotoxic compounds, 5-6 cytotoxic compounds, or 2-5 cytotoxic compounds and each cytotoxic compound on the conjugate is the same.

In certain embodiments, a composition of the ADC of the present invention has an average drug to antibody ratio (DAR) of 1.0 to 5.0, 1.5 to 4.0, 2.0 to 3.5, 2.5 to 3.1, or 2.7 to 2.9. In one embodiment, the DAR is 2.8.

In one embodiment, the cytotoxic compound of the present invention is represented by the follo ing formula:

or a pharmaceutically acceptable salt thereof, wherein the antibody is an anti-CD 19 antibody, r_c is 1 or 2, and Y is -S0₃H. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt or a potassium salt. In certain embodiments, Y is -S0₃Na or -S0₃K. In certain embodiments, Y is -S0₃Na. The cytotoxic compounds and the antibody-drug conjugates shown above can be prepared using the methods and procedures disclosed in International Patent Application Publication No. WO 2017/004025, which is incorporated herein by reference in its entirety. In certain embodiments, for the conjugates disclosed herein, the anti-CD19 antibody binds specifically to CD 19 and comprises six complementary determining regions (CDRs) including: i) a light chain CDRl comprising SASSGVNYMH (SEQ ID NO: 1); a light chain CDR2 comprising DTSKLAS (SEQ ID NO: 2); and a light chain CDR3 comprising

HQRGSYT (SEQ ID NO: 3); and ii) a heavy chain CDRl comprising SNWMH (SEQ ID NO:4); a heavy chain CDR2 comprising EIDPSDSYTN (SEQ ID NO:5); and a heavy chain CDR3 comprising GSNPYYYAMDY (SEQ ID NO:6).

In certain embodiments, the anti-CD19 antibody in the conjugates of the present invention comprises a light chain, wherein the sequence of the light chain has at least 60%, at least 75%, at least 85%, at least 95% or at least 99% identity with the sequence displayed in SEQ ID NO: 7.

Glu He val Leu Thr Gin Ser Pro Ala lie Met ser Ala Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Asn Tyr Met

20 25 30

His Trp Tyr Gin Gin Lys Pro Gly Thr Ser Pro Arg Arg Trp lie Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Ser Leu Thr lie Ser Ser Met Glu Pro Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gin Arg Gly Ser Tyr Thr Phe Gly

85 90 95

Gly Gly Thr Lys Leu Glu lie Lys Arg Thr val Ala Ala Pro Ser Val

100 105 110

Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser

115 120 125

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin

130 135 140

Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Val

145 150 155 160

Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

165 170 175

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

180 185 190

Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

195 200 205

Gly Glu Cys (SEQ ID NO: 7)

210 In certain embodiments, the anti-CD19 antibody in the conjugates of the present invention comprises a heavy chain, wherein the sequence of the heavy chain has at least 60%, at least 75%, at least 85%, at least 95% or at least 99% identity with the sequence displayed in SEQ ID NO: 8.

Gin Val Gin Leu Val Gin Pro Gly Ala Gl u val Val Lys Pro Gly Ala

1 5 10 15

Ser val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr ser Asn

20 25 30

Trp Met His Trp val Lys Gin Ala Pro Gly Gin Gly Leu Gl u Trp lie

35 40 45

Gly Gl u lie Asp Pro ser Asp Ser Tyr Thr Asn Tyr Asn Gin Asn Phe

50 55 60

Gin Gly Lys Ala Lys Leu Thr val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

MetGlu Val Ser Ser Leu Arg ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gin

100 105 110

Gly Thr Ser val Thr Val Ser Ser Ala ser Thr Lys Gly Pro Ser val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu val Lys Asp Tyr Phe Pro Gl u Pro val Thr val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr ser Gly val His Thr Phe Pro Ala val

165 170 175

Leu Gin Ser Ser Gly Leu Tyr Ser Leu ser ser valval Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys val Asp Lys Lys val Gl u Pro Lys ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gl u Leu Leu Gly Gly

225 230 235 240

Pro Ser val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie

245 250 255

Ser Arg Thr Pro Gl u val Thr Cys Val Val Val Asp val ser Hi s Gl u

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly val Glu Val His

275 280 285 Asn Ala Lys Thr Lys Pro Arg Gl u Glu Gin Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu

325 330 335

Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Gl u Leu Thr Lys Asn Gin Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gl n Gin Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys (SEQ ID NO: 8)

435

In certain embodiments, the anti-CD19 antibody in the conjugates of the present invention is the humanized antibody huB4 described in Roguska et al. {Proc. Natl. Acad. Sci. USA, 91: 969-973, 1994). The antibody huB4 comprises a light chain having the sequence of SEQ ID NO: 7 and a heavy chain having the sequence of SEQ ID NO: 8.

Compositions and Methods of Use

The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) that can be used in the preparation of unit dosage forms. Such

compositions comprise a therapeutically effective amount of the conjugates of the present invention and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term

"carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.

Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with a conjugate of the present invention, alone or with such

pharmaceutically acceptable carrier. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The present invention provides kits that can be used in the above methods. A kit can comprise any of the conjugates of the present invention.

The conjugates and compositions of the present invention may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder by administering to a subject a therapeutically effective amount a conjugate of the invention. In a preferred aspect, such compositions are substantially purified (i.e. , substantially free from substances that limit its effect or produce undesired side effects). In a specific embodiment, the subject is an animal, preferably a mammal such as non-primate (e.g. , bovine, equine, feline, canine, rodent, etc.) or a primate (e.g. , monkey such as, a cynomolgus monkey, human, etc.) In a preferred embodiment, the subject is a human.

In certain embodiments, the conjugates may be administered to a subject, in a pharmaceutically acceptable dosage form.

Methods of administering a conjugate of the invention include, but are not limited to, parenteral administration (e.g. , intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g. , intranasal and oral routes). In a specific embodiment, the conjugates of the present invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, and may be administered together with other biologically active agents. Administration can be systemic or local. As used herein, an "therapeutically effective amount" of a pharmaceutical composition is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results such as decreasing symptoms of cancer (e.g., the proliferation, of cancer cells, tumor presence, tumor metastases, etc.), thereby increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication such as via targeting and/or internalization, delaying the progression of the disease, and/ or prolonging survival of individuals.

In one aspect, the present invention provides a method of treating a cancer in a mammal (e.g. a human) comprising administering to the mammal a therapeutically effective amount of any one or more conjugates described herein. In certain instances, the cancer is a B-cell malignancy. In certain instances, the cancer is a leukemia or lymphoma. In some instances, the cancer is selected from the group consisting of B-cell lymphomas including non-Hodgkin's lymphoma (NHL), precursor B-cell lymphoblastic leukemia/lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B- cell lymphoma (MALT type, nodal type, and splenic marginal zone lymphoma (SMZL), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), activated B cell like diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B cell like diffuse B-cell lymphoma (GCB-DLBCL), Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, anaplastic large-cell lymphoma (ALCL), and primary mediastinal large B-cell lymphoma (PMBCL) and B-cell acute lymphoblastic leukemia (B-ALL).

In certain embodiments, the cancer is B-ALL.

In certain instances, the cancer is a T-cell malignancy.

In certain embodiments, the cancer is NHL, DLBCL, FL or MCL. In certain embodiments, the cancer is ABC-DLBCL or GCB-DLBCL.

The therapeutic applications of the present invention can be also practiced in vitro and ex vivo. Production of Antibody-Drug Conjugates

In order to link the cytotoxic compounds of the present invention to the anti-CD 19 antibody, the cytotoxic compound may comprise a linking moiety with a reactive group bonded thereto. In one embodiment, the sulfo-SPDB linker can first react with the cytotoxic compound to provide the compound bearing a linking moiety with one reactive group bonded thereto (i.e., drug-linker compound), which can then react with the antibody. Alternatively, one end of the sulfo-SPDB linker can first react with the antibody to provide the antibody bearing a linking moiety with one reactive group bonded thereto, which can then react with a cytotoxic compound. In certain embodiments, the compounds and the conjugate of the present invention can be prepared according to procedures described in U.S. Patent Nos. 8,765,740 and 9,353,127, incorporated herein by reference.

All references cited herein and in the examples that follow are expressly incorporated by reference in their entireties.

EXAMPLES

The invention will now be illustrated by reference to non-limiting examples. Unless otherwise stated, all percentages, ratios, parts, etc. are by weight. All reagents were purchased from the Aldrich Chemical Co., New Jersey, or other commercial sources.

Example 1. In vitro activity of huB4-DGN462 in lymphoma cell lines

Methods. The humanized anti-CD 19 antibody, huB4, was conjugated to DGN462 via a cleavable disulfide linker, sulfo-SPDB. In vitro activity of the huB4-DGN462 ADC or the unconjugated DGN462SMe toxin was evaluated in 54 lymphoma cell lines (27 diffuse large B cell lymphomas (DLBCL); 10 mantle cell lymphomas; 6 marginal zone lymphomas; 5 anaplastic large T-cell lymphomas; 6 others). Cell proliferation/viability after 72h of exposure was measured using a MTT assay. Apoptosis activation was defined by at least 1.5- fold increase in caspase 3/7 signal activation in respect to controls using the Promega ApoTox-Glo Triplex Assay. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > 121, P<0.05, FDR<0.25) and limma t-test (FC> 11.21; P< 0.05; top 200 up and top 200 down).

Results. As shown in FIG. 1, huB4-DGN462 was cytotoxic against a broad panel of 54 B cell lymphoma cell lines (median IC50 100 pM; 95%CI, 38-214). The cytotoxic activity was not limited by P53, BCL2, MYC or CDKN2A status, or associated with DLBCL cell of origin. Consistent with overall lower CD19 expression, huB4-DGN462 was significantly (p-value=0.007) less active in eight T cell-derived lymphomas (median IC₅₀ of 1.75 nM (95%CI, 0.5-5.75)) than in B cell lymphomas. huB4-DGN462 induced caspase 3/7 activation in 48/54 cell lines (89%) consistent with an apoptotic mechanism of action.

Example 2. In vivo efficacy in mice xenograft models

In vivo efficacy of huB4-DGN462 was evaluated in two CD19-expressing xenograft tumor models. As shown in FIGs. 2 and 3, huB4-DGN462 demonstrated compelling antitumor activity after a single intravenous dose in two diffuse large B-cell lymphoma cell line xenograft models: DoHH2 (a subcutaneous model) and Farage (a disseminated model). Data collection and analysis for all disseminated models

The mice were weighed twice a week and were monitored for clinical signs throughout the duration of the study. The measured end-point was survival. Animals were euthanized when hind leg paralysis was present, body weight decreased by >20% of pre- treatment weight, a visible tumor appeared, or any signs of distress were visible.

Spontaneous deaths were recorded when they were discovered.

For disseminated models, Tumor Growth Delay is calculated as T-C, where T is the median survival time (in days) of a treated group and C is the median survival time (in days) of the vehicle control group. The Percent Increased Life Span (%ILS) for disseminated models is calculated using the following formula: %ILS = (T-C) / C x 100%. Anti-tumor activity was evaluated as per NCI standards for disseminated models: ILS > 25% is minimum active, ILS > 40% is active, and ILS > 50% is highly active.

Data collection and analysis for all subcutaneous models

The mice were weighed twice a week and were monitored for clinical signs throughout the duration of the study. Animals were euthanized when hind leg paralysis was present, body weight decreased by >20% of pre-treatment weight, tumor ulceration occurred, or when any signs of distress were visible.

Tumor volumes were measured one to two times weekly in three dimensions using a caliper. The tumor volume was expressed in mm3 using the formula V = Length x Width x Height x ½ (Tomayko and Reynolds, Cancer Chemother. Pharmacol. 24: 148-54 (1989)). Activity was assessed as described in Bissery et al., Cancer Res. 51: 4845-52 (1991).

Tumor Growth Inhibition (T/C Value) was also assessed using the following formula: T/C (%) = (Median tumor volume of the treated / Median tumor volume of the control) x 100%. Tumor volume was determined simultaneously for the treated (T) and the vehicle control (C) groups when tumor volume of the vehicle control reached a predetermined size (Bissery et al., Cancer Res. 51: 4845-52 (1991). The daily median tumor volume of each treated group was determined, including tumor-free mice (0 mm ). According to NCI standards, a T/C < 42% is the minimum level of anti-tumor activity. A T/C < 10% is considered a high antitumor activity level. A mouse was considered to have a partial regression (PR) when tumor volume was reduced by 50%, or greater and a complete tumor regression (CR when no palpable tumor could be detected. 1) DoHH2 Model

CB 17-SCID mice were subcutaneously inoculated with lxlO⁷ DOHH2 cells (GCB- DLBCL). Mice were randomized into study groups (n=6) when tumor volume reached ~ 100mm and the following day mice received single IV injections of either vehicle, non- targeted control (IgGl-DGN462) or huB4-DGN462 as indicated. Data regarding tumor growth and drug activity are reported. Tumor Growth Inhibition (% T/C) = (Median tumor volume of test article-treated group) / (Median tumor volume of vehicle-treated group) x 100%.

As shown in FIG. 2 and Table 1, huB4-DGN462 resulted in a significant, dose- dependent tumor growth delay and survival benefit at 1.7 mg Ab/kg compared to a non- targeted control DGN462 ADC.

Table 1.

2) Farage Model

CB 17-SCID mice were injected IV with lxlO⁷ FARAGE cells (GCB-DLBCL) on Day 0 and randomized into study groups (n=8) on Day 6. On Day 7, mice received a single IV injection of either vehicle, non-targeted control (IgGl-DGN462) or huB4-DGN462 as indicated. Data regarding tumor growth delay, survival and drug activity are reported. Tumor Growth Delay (T-C), where T = median survival of the ADC-treated group, and C = median survival of the vehicle-treated group. Increased Life Span (% ILS) = (T-C)/C X100%.

As shown in FIG. 3 and Table 2, a significant dose-dependent increase in survival was observed in mice treated with as low as 0.17 mg Ab/kg of huB4-DGN462. At 1.7 mg Ab/kg, the life span was increased >400% compared to untreated mice.

Table 2.

Example 3. In vitro cytotoxicity of huB4-DGN462 conjugate in B-NHL and B-ALL cell lines

The in vitro cytotoxicity of huB4-DGN462 conjugate and huB4-DGN462 conjugate blocked with excess non-conjugated antibody against a panel of B-NHL and B-ALL cell lines were compared to in vitro cytotoxicity of a conjugate of the huB4 antibody linked to a maytansinoid payload (huB4-SPDB-DM4) and huB4-SPDB-DM4 blocked with excess non- conjugated antibody. Specifically, 2000 cells /well were plated in 96-well plates and conjugates added. Conjugates were diluted into the culture medium using 3-fold dilution series and ΙΟΟμί were added per well. Control wells containing cells but lacking conjugate, as well as wells contained medium only, were included in each assay plate. Assays were performed in triplicated for each data point. The plates were incubated at 37°C in a humidified 5% C0₂ incubator for 5 days. Then the relative number of viable cells in each well was determined using the WST-8 based Cell Counting Kit-8 (Dojindo Molecular Technologies). The surviving fraction of cells in each well was calculated by first correcting for the medium background absorbance, and then dividing each value by the average of the values in the control wells (non-treated cells). The percentage of surviving cells was plotted against conjugate concentration and the EC50 of activity was calculated using a nonlinear regression analysis (GraphPad Prism 7.0).

Surprisingly, as shown in FIG. 4 and Table 3, huB4-DGN462 conjugate demonstrated a greater than 2-log superior in vitro cytotoxicity activity in all B-ALL cell lines tested as compared to the huB4-SPDB-DM4 conjugate.

Table 3.

huB4-SPDB-DM4 3517

TOM-1

huB4-DGN462 16

Example 4: In Vivo Efficacy huB4-s-SPDB-DGN462, huB4-DGN549 in DoHH2

Subcutaneous Model

To test the efficacy of huB4-s-SPDB-DGN462 and huB4-DGN549 for the ability to decrease tumor burden in vivo, a subcutaneous tumor model was used as described in the protocol below.

Female CB.17 SCID mice were each inoculated with 10x106 DoHH2 cells, a human DLBCL cell line, in 100 μΐ_^ serum free medium, subcutaneously in the right flank. On day 10 post-DoHH2 inoculation, mice were randomized into the study groups. On day 10 post- DoHH2 inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 5.8 μg/kg (by DGN462; 0.3 mg/kg by huB4) huB4-s-SPDB-DGN462, 11.7 μg/kg (by DGN462; 0.6 mg/kg by huB4) huB4-s-SPDB-DGN462, 23.4 μg/kg (by DGN462; 1.2 mg/kg by huB4) huB4-s-SPDB-DGN462, 9.5 μg/kg (by DGN462; 0.6 mg/kg by chKTI) chKTI-s-SPDB-DGN462 control conjugate, 19 μg/kg (by DGN462; 1.2 mg/kg by chKTI) chKTI-s-SPDB-DGN462 control conjugate, 1.3 μg/kg (by DGN549; 0.075 mg/kg by huB4) huB4-DGN549, 2.7 μg/kg (by DGN549; 0.15 mg/kg by huB4) huB4-DGN549, 5.3 μg/kg (by DGN549; 0.3 mg/kg by huB4) huB4-DGN549, 2.4 μg/kg (by DGN549; 0.15 mg/kg by chKTI) chKTI-DGN549 control conjugate, 4.7 μg/kg (by DGN549; 0.3 mg/kg by chKTI) chKTI-DGN549 control conjugate, or 106.3 μg/kg (by DM4; 5 mg/kg by huB4) huB4-s- SPDB-DM4 (Coltuximab ravtansine).

The results are represented in Table 4 and in FIGS. 5 (with huB4-s-SPDB-DM4 or Coltuximab ravtansine).

The 0.3 and 0.6 mg/kg (by huB4) doses of huB4-s-SPBD-DGN462 were active, generating a 32 %T/C and a 24 %T/C, respectively, and 0/6 CRs each. The 1.2 mg/kg (by huB4) dose of huB4-s-SPDB-DGN462 was highly active, generating a 4 %T/C and 2/6 CRs. In contrast, the 0.6 and 1.2 mg/kg (by huB4) doses of the chKTI-s-SPDB-DGN462 control conjugate were inactive, generating a 70 %T/C and a 47 %T/C, respectively, and 0/6 CRs each, demonstrating that the activity of huB4-s-SPDB-DGN462 was CD19-dependent. The 0.075 mg/kg (by huB4) dose of huB4-DGN549 was active, generating a 36 %T/C and 0/6 CRs, while the 0.15 and 0.3 mg/kg (by huB4) doses of huB4-DGN549 were both highly active, generating an 8 %T/C and a 0 %T/C, respectively, and 0/6 CRs and 5/6 CRs, respectively. In contrast, the 0.15 mg/kg (by huB4) dose of chKTI-DGN549 control conjugate was inactive, generating a 74 %T/C and 0/6 CRs. While the 0.3 mg/kg (by huB4) dose of chKTI-DGN549 was active, generating a 25 %T/C, it resulted in 0/6 CRs. Together, the results from chKTI-DGN549 demonstrate the CD19-dependent activity of huB4-

DGN549. The 5 mg/kg (by huB4) dose of huB4-s-SPDB-DM4 (Coltuximab ravtansine) active, generating an 11 %T/C and 2/6 CRs.

Table 4.

Example 5: In Vivo Efficacy huB4-s-SPDB-DGN462, huB4-DGN549 in OCI-Lyl8 Subcutaneous Model

Female CB.17 SCID mice were each inoculated with 10x106 OCI-Lyl8 cells, a human DLBCL cell line, in 100 μΐ_^ Matrigel/ serum free medium, subcutaneously in the right flank. On day 12 post-OCI-Lyl8 inoculation, mice were randomized into the study groups. On day 13 post-OCI-Lyl8 inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 5.8 μg/kg (by DGN462; 0.3 mg/kg by huB4) huB4-s-SPDB- DGN462, 11.7 μg/kg (by DGN462; 0.6 mg/kg by huB4) huB4-s-SPDB-DGN462, 23.4 μg/kg (by DGN462; 1.2 mg/kg by huB4) huB4-s-SPDB-DGN462, 9.5 μg/kg (by DGN462; 0.6 mg/kg by chKTI) chKTI-s-SPDB-DGN462 control conjugate, 19 μg/kg (by DGN462; 1.2 mg/kg by chKTI) chKTI-s-SPDB-DGN462 control conjugate, 1.3 μg/kg (by DGN549; 0.075 mg/kg by huB4) huB4-DGN549, 2.7 μg/kg (by DGN549; 0.15 mg/kg by huB4) huB4- DGN549, 5.3 μg/kg (by DGN549; 0.3 mg/kg by huB4) huB4-DGN549, 2.4 μg/kg (by DGN549; 0.15 mg/kg by chKTI) chKTI-DGN549 control conjugate, 4.7 μg/kg (by DGN549; 0.3 mg/kg by chKTI) chKTI-DGN549 control conjugate, or 106.3 μg/kg (by DM4; 5 mg/kg by huB4) huB4-s-SPDB-DM4 (Coltuximab Ravtansine).

The results are represented in Table 5 and in FIGS. 6 (with huB4-s-SPDB-DM4 or Coltuximab ravtansine).

The 0.3 mg/kg (by huB4) dose of huB4-s-SPDB-DGN462 was inactive, generating a 53 %T/C and 0/6 CRs. The 0.6 and 1.2 mg/kg (by huB4) doses of huB4-s-SPBD-DGN462 were active, generating a 21 %T/C and a 19 %T/C, respectively, and 0/6 CRs and 1/6 CRs, respectively. In contrast, the 0.6 and 1.2 mg/kg (by huB4) doses of the chKTI-s-SPDB- DGN462 control conjugate were inactive, generating a 69 %T/C and an 81 %T/C, respectively, and 0/6 CRs each, demonstrating that the activity of huB4-s-SPDB-DGN462 was CD19-dependent. The 0.075 mg/kg (by huB4) dose of huB4-DGN549 was inactive, generating a 46 %T/C and 1/6 CRs, while the 0.15 and 0.3 mg/kg (by huB4) doses of huB4- DGN549 were both active, generating a 14 %T/C and an 18 %T/C, respectively, and 0/6 CRs and 2/6 CRs, respectively. In contrast, the 0.15 and 0.3 mg/kg (by huB4) doses of chKTI- DGN549 control conjugate were inactive, generating a 68 %T/C and a 44% T/C,

respectively, and 0/6 CRs each, demonstrating the CD19-dependent activity of huB4- DGN549. The 5 mg/kg (by huB4) dose of huB4-s-SPDB-DM4 (Coltuximab ravtansine) was highly active, generating a 5 %T/C and 1/6 CRs.

Table 5.

huB4-s-SPDB-DGN462 1.2 23.4 19 1/6 1/6 Active chKTI-s-SPDB-DGN462 0.6 9.5 69 0/6 0/6 Inactive chKTI-s-SPDB-DGN462 1.2 19 81 0/6 0/6 Inactive huB4-DGN549 0.075 1.3 46 1/6 1/6 Inactive huB4-DGN549 0.15 2.7 14 0/6 0/6 Active huB4-DGN549 0.3 5.3 18 3/6 2/6 Active chKTI-DGN549 0.15 2.4 68 0/6 0/6 Inactive chKTI-DGN549 0.3 4.7 44 0/6 0/6 Inactive huB4-s-SPDB-DM4, 5 106.3 5 4/6 1/6 Highly Active (Coltuximab ravtansine)

Claims

CLAIMS We claim:

1. An anti-CD 19 antibody-drug conjugate represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein r is an integer from 1 to 10, Y is - S0₃H, or a pharmaceutically acceptable salt thereof, and the antibody is an anti-CD19 antibody that specifically binds to a CD 19 antigen and comprises:

i) a light chain CDRl comprising SASSGVNYMH (SEQ ID NO: 1); a light chain CDR2 comprising DTSKLAS (SEQ ID NO: 2); and a light chain CDR3 comprising HQRGSYT (SEQ ID NO: 3); and

ii) a heavy chain CDRl comprising SNWMH (SEQ ID NO: 4); a heavy chain CDR2 comprising EIDPSDSYTN (SEQ ID NO: 5); and a heavy chain CDR3 comprising

GSNPYYYAMDY (SEQ ID NO: 6).

2. la:

i) a light chain CDRl comprising SASSGVNYMH (SEQ ID NO: 1); a light chain

GSNPYYYAMDY (SEQ ID NO: 6). -CD 19 antibody-drug conjugate represented by the following formula:

HQRGSYT

(SEQ ID NO: 3); and

GSNPYYYAMDY (SEQ ID NO: 6).

4. The conjugate of any one of claims 1-3, wherein the anti-CD19 antibody is huB4 antibody comprising a light chain having the sequence of SEQ ID NO: 7 and a heavy chain having the sequence of SEQ ID NO: 8.

5. The conjugate of any one of claims 1-4, wherein the pharmaceutically acceptable salt is a sodium salt or a potassium salt.

6. The conjugate of any one of claims 1-5, wherein Y is -S0₃Na.

7. A pharmaceutical composition comprising the conjugate of any one of claims 1-6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

8. A method of treating a cancer in a mammal in need thereof, comprising administering to said mammal a therapeutically effective amount of the conjugate of any one of claims 1-6 or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the cancer is a B-cell malignancy.

10. The method of claim 8, wherein the cancer is a leukemia or lymphoma.

11. The method of claim 8, wherein the cancer is selected from the group consisting of B- cell lymphomas including non-Hodgkin's lymphoma (NHL), precursor B-cell lymphoblastic leukemia/lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, B- cell acute lymphoblastic leukemia (B-ALL), lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal type, and splenic marginal zone lymphoma (SMZL), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), activated B cell like diffuse large B-cell lymphoma (ABC- DLBCL), germinal center B cell like diffuse B-cell lymphoma (GCB-DLBCL), Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macro globulinemia, anaplastic large-cell lymphoma (ALCL), and primary mediastinal large B-cell lymphoma (PMBCL).

12. The method of claim 8, wherein the cancer is non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) or mantle cell lymphoma (MCL).

13. The method of claim 8, wherein the cancer is activated B cell like diffuse large B-cell lymphoma (ABC-DLBCL) or germinal center B cell like diffuse B-cell lymphoma (GCB- DLBCL).

14. The method of claim 8, wherein the cancer is B-cell acute lymphoblastic leukemia (B-ALL).