Methods and pharmaceutical composition for the treatment of adenoid cystic carcinoma

Abstract

Methods for using antibodies, antigen-binding antibody fragments, immunoconjugates and pharmaceutical compositions for the treatment of adenoid cystic carcinoma, and pharmaceutical compositions containing immunoconjugates.

Description

METHODS AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OFADENOID CYSTIC CARCINOMACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S. Provisional application 63 / 567,381, filed on March 19, 2024, the contents of which is incorporated herein by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The sequence listing in ST.26 XML format entitled BIAT1038WOSequenceListing.xml, created on March 12, 2025, comprising 178,713 bytes, prepared according to 37 CFR 1.822 to 1.824, submitted concurrently with the filing of this application, is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] This disclosure relates to methods and pharmaceutical compositions for the treatment of adenoid cystic carcinoma (ACC) using an antibody drug conjugate comprising a conditionally active anti-AXL antibody or antigen binding antibody fragment.BACKGROUD OF THE DISCLOSURE

[0004] Adenoid cystic carcinoma (ACC) is the most common form of salivary gland carcinoma, however, in terms of the general population it is relatively rare with a U.S. incidence rate of 0.35 per 100,000 persons (Boyle et al., J. Clin. Oncol, vol. 38 (suppl. 15), pp el3600, 2020). ACC is considered to be an aggressive form of cancer since approximately 50% of patients will experience reoccurrence and distant metastasis following initial treatment (Spiro, Am. J. Surg., vol. 174, pp495-498, 1977; Jang, et al., Anticancer Res., vol. 37, pp 3045-3052, 2017; Ellington, et al., Cancer, vol. 118, pp 4444- 4451, 2012). Currently, there are no FDA approved drugs for the treatment of ACC (Wagner, et al., Critical Rev. Oncology, Hematology, vol.181, 103886, 2023).

[0005] ACC exhibits two molecular subtypes, type I adenoid cystic carcinoma (ACC-I) and type I adenoid cystic carcinoma (ACC-II). ACC-I is the more aggressive subtype with a median overall survival rate of 3.4 years and represent about 30% of all ACC cases. The remainder of cases are of ACC-II which is slower progressing and has a median overall survival rate of 23.2 years. Proteomicanalysis has shown that AXL gene and protein expression are significantly increased in the ACC-II subtype (Ferrarotto, et al., Clin. Cancer Res., vol. 23, pp 852-864, 2021).

[0006] AXL protein (also known as Ark, UFO, Tyro-7) is a tyrosine kinase receptor in the Tyro- 3 family of kinases. The Tyro-3 kinase receptors are characterized by a combination of two immunoglobin-like domains, dual fibronectin type III repeats in the extracellular region and a cytoplasmic kinase domain. The ligands for Tyro-3 kinase receptors are Gas6 (growth-arrest-specific 6) and protein S, two vitamin-K dependent proteins that show 43% amino acid sequence identity and share similar domain structures. Each protein has an N-terminal Gia domain containing 11 g- carb oxy glutamic acid residues, followed by four epidermal growth factor (EGF)-like modules, and a C -terminal sex hormone-binding globulin (SHBG)-like structure consisting of two tandem laminin G domains. The SHBG domain is both necessary and sufficient for Tyro-3 kinase receptor binding and activation, whereas the Gia domain binds the negatively charged membrane phospholipids and plays an important role in Tyro-3 kinase-mediated phagocytosis of apoptotic cells.

[0007] AXL is weakly expressed in a range of normal tissues, including brain, heart, skeletal muscle, organ capsules and connective tissues of several other organs, and in monocytes, but not lymphocytes. Akt phosphorylation induced by AXL has been described in survival of fibroblasts (Goruppi et al., Mol. Cell. Biol., vol. 17, pp. 4442-4453 1997), endothelial cells (Hasanbasic et al., 4 / 7 / J Physiol Heart Circ Physiol, vol. 287, H1207-H1213, 2004), vascular smooth muscle cells (Melaragno et al., J. Mol. Cell. Cardiol., vol. 37, pp. 881-887, 2004) and neurons (Allen et al., Mol. Endocrinol., vol. 13, pp. 191- 201, 1999). Furthermore, AXL plays a role in cell-adhesion and chemotaxis because AXL knockout animals display impaired platelet aggregate stabilization and thrombus formation as a result of reduced activation of the platelet integrin lib 3.

[0008] AXL over expression in cancer is generally associated with a poor prognosis (Zucca, et al., Urol OncoL, vol. 36, ppl lel3-lle21, 2018; Liu, et al., PLoS One, vol. 11, p e0154767). AXL has been identified to be related to tumor metastasis because AXL is upregulated in aggressive breast cancer cell lines compared to non-invasive cells. In vitro, AXL activity was found to be required for migration and invasion. This activity can be inhibited by antibody treatment (see e.g. WO 2004 / 008147). Similarly, abrogation of AXL activity in vivo, either via expression of a dominant negative version of AXL (Vajkoczy, P., et al., Proc. Natl. Acad. Science U.S.A., vol. 103, pp. 5799-5804, 2005) or by siRNA mediated downregulation of AXL (Holland et al., Cancer Res., vol. 65, pp. 9294-9303, 2005) prevented subcutaneous and orthotopic cell growth in murine xenograft experiments.

[0009] Accordingly, anti-AXL monoclonal antibodies have been described for use in the treatment of cancers. For example, publications relating to anti-AXL antibodies include WO 2009 / 063965, WO 2009 / 062690, WO 2011 / 014457, US 2014 / 0227283, and U.S. Patent No. 8,853,369. US 2014 / 0227283 discloses monoclonal anti-AXL antibodies and uses thereof in diagnostic and therapeutic methods. WO 2009 / 062690 discloses antibodies that bind to the extracellular domain of the AXL protein and can at least partially inhibit AXL activity.

[0010] These monoclonal anti-AXL antibodies will bind to AXL at any location of a patient’s body with similar affinity, including at locations of the tumors they are intended to treat. The binding of such antibodies to AXL in non-tumor environments is expected to have an adverse effect on the normal functioning of AXL in these environments and thus may cause significant side-effects. The present disclosure provides conditionally active anti-AXL antibodies and antibody fragments that have a higher binding affinity to AXL in a tumor microenvironment in comparison with their binding affinity to AXL in a non-tumor environment. The anti-AXL antibodies and antigen binding antibody fragments of the present disclosure are expected to have comparable or greater anti-cancer efficacy with reduced side-effects, in comparison with the monoclonal anti-AXL antibodies known in the art. This may also permit administration of higher dosages of the anti-AXL antibodies and antigen binding antibody fragments or more frequent treatment, thus providing a more effective therapeutic option.

[0011] Given the aggressive nature of ACC and the lack of FDA approved drugs, there is an urgent need for an effective systemic therapy to treat ACC (Wagner, et al., Critical Rev. Oncology, Hematology, vol. 181, 103886, 2023).SUMMARY OF THE DISCLOSURE

[0012] Provided herein is a method of treating adenoid cystic carcinoma, comprising administering to a subject having adenoid cystic carcinoma a pharmaceutical composition comprising AbCAB-(linker- D)nand a pharmaceutically acceptable carrier at a dose of 1.8 mg / kg once every two weeks, wherein AbCAB is a conditionally active anti-AXL antibody or antigen binding antibody fragment, linker is a cleavable linker, D is an anti-cancer agent, and n is an integer from 1 to 4. In certain embodiments, the conditionally active anti-AXL antibody or antigen binding antibody fragments is a pH dependent conditionally active antibody or antigen binding antibody fragment.

[0013] Also provided herein is a pharmaceutical composition for administration to a patient having adenoid cystic carcinoma, said pharmaceutical composition comprising AbCAB-(linker-D)nand a pharmaceutically acceptable carrier comprising a dose of 1.8 mg / kg, wherein AbCAB is aconditionally active anti-AXL antibody or antigen binding antibody fragment, linker is a cleavable linker, D is an anti-cancer agent, and n is an integer from 1 to 4.

[0014] The conditionally active anti-AXL antibody or antigen binding antibody fragment of each of the foregoing embodiments may include six complementarity determining regions Hl, H2, H3, LI, L2 and L3, wherein: the Hl amino acid sequence is X1GX2X3MX4 (SEQ ID NO: 1): whereinXi is T or A or W,X2 is H or A,X3 is T or I, andXHs N or I; the H2 amino acid sequence is LIKXsSNGGTXeYNQKFKG (SEQ ID NO: 2): whereinX5 is P or N, andX()is S or I or T; and the H3 amino acid sequence is GX7X8X9X10X11X12X13X14DYX15X16 (SEQ ID NO: 3): whereinX? is H or D or E or P or R or W,Xg is Y orN,X9 is E or A or D or F or G or H or I or L or M or N or R or V or Y,X10 is S or D or M or N or Q,Xu is Y or C or E or P,X12 is F or E or N or S or T or V,X13 is A or D or G or L or Y,X14 is M or E or F,X15 is W or A or D or H or L or N or P or R or T, andXi6 is G or H, the LI amino acid sequence is KASQDX17X18SX19VX20 (SEQ ID NO: 4): whereinX17 is V or D or G or N or W,Xis is S or V,X19 is A or L or M, andX20 is A or D or N or Q; the L2 amino acid sequence is X21X22X23TRX24T (SEQ ID NO: 5): whereinX21 is W or F,X22 is A or I or N or P or Q,X23 is S or D, andX24 is H or D; and the L3 amino acid sequence is QEX25X26SX27X28X29X30 (SEQ ID NO: 6): whereinX25 is H or C or F or I or L or Q or S or T or V or Y,X26 is F or C or D or E or G or N or S,X27 is T or C or P,X28 is P or A or C or D or E or H or K or S or T or V or W,X29 is L or G or R, andX30 is T or I or R; with the proviso that the antibody or antigen binding antibody fragment cannot have a combination of the following six CDRs:Hl = TGHTMN (SEQ ID NO: 117),H2 = LIKPSNGGTSYNQKFKG (SEQ ID NO: 118),H3 = GHYESYFAMDYWG (SEQ ID NO: 119),LI = KASQDVSSAVA (SEQ ID NO: 120), L2 = WASTRHT (SEQ ID NO: 121), and L3 = QEHFSTPLT (SEQ ID NO: 122).

[0015] The conditionally active anti-AXL antibody or antigen-binding antibody fragment of any of the foregoing embodiments may have up to three substitutions in the CDRs Hl, H2 and H3, relative to the HC-WT shown in Fig. 1A and up to five substitutions in the CDRs LI, L2 and L3, relative to the LC-WT shown in Fig. IB. This includes conditionally active anti-AXL antibodies or antigen binding antibody fragments with one substitution in the CDRs Hl, H2 and H3, relative to the HC-WT shown in Fig. 1A, conditionally active anti-AXL antibodies or antigen binding antibody fragments with one substitution in the CDRs LI, L2 and L3, relative to the LC-WT shown in Fig. IB, and conditionally active anti-AXL antibodies or antigen binding antibody fragments with one substitution in the CDRs Hl, H2 and H3, and one substitution in the CDRs LI, L2 and L3, relative to the HC-WT shown in Fig. 1A and the LC-WT shown in Fig IB, respectively. The possible combinations of the CDRs Hl, H2 and H3 are shown in Fig. 1A and the possible combinations of the CDRs LI, L2 and L3 are shown in Fig. IB. In any of the foregoing embodiments, the specific combination of the CDRs in the HC-WT shown in Fig. 1A with the CDRs of the LC-WT shown in Fig. IB may be excluded from the possible combinations of the present invention.

[0016] One specific embodiment of the foregoing antibodies and antigen binding antibody fragments is a conditionally active anti-AXL antibody or antigen binding antibody fragment containing six complementarity determining regions Hl, H2, H3, LI, L2 and L3 in which Xi is W, X2 is A, X3 is T, X4is N, X5is P, X6is S, X7is H, X8is Y, X9is E, X10 is S, Xu is Y, X12 is E, X13 is A, Xi4is M, X15 is W, Xis is G, X17 is V, Xis is V, X19 is A, X20 is A, X21 is W, X22 is Q, X23 is D, X24is H, X25is H, X26is F, X27 is P, X2s is P, X29 is L and X30 is T.

[0017] Another specific embodiment of the foregoing antibodies and antigen binding antibody fragments is a conditionally active anti-AXL antibody or antigen binding antibody fragment containing a heavy chain variable region comprising an amino acid sequence of selected from amino acid sequence of SEQ ID NOs: 129, 131, 133, 135, 136, and 137 and a light chain variable region comprising an amino acid sequence of selected from amino acid sequences of SEQ ID NOs: 48, 128, 132, 134, and 138.

[0018] Another specific embodiment of the foregoing antibodies and antigen binding antibody fragments is a conditionally active anti-AXL antibody or antigen binding antibody fragment that comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 128 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 129, a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 131 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 132, a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 128, a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 136 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:48, or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 137 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 138.

[0019] Another specific embodiment of the foregoing antibodies and antigen binding antibody fragments is a conditionally active anti-AXL antibody or antigen binding antibody fragment containing six complementarity determining regions Hl, H2, H3, LI, L2 and L3 in which Hl is the amino acid sequence WGATMN (SEQ ID NO: 123), H2 is the amino acid sequence LIKPSNGGTSYNQKFKG (SEQ ID NO: 118), H3 is the amino acid sequence GHYESYEAMDYWG (SEQ ID NO: 124), LI is the amino acid sequence KASQDVVSAVA (SEQ ID NO: 125), L2 is theamino acid sequence WQDTRHT (SEQ ID NO: 126) and L3 is the amino acid sequence QEHFSPPLT (SEQ ID NO: 127).

[0020] In another specific embodiment of the foregoing antibodies and antibody fragments, the present disclosure relates to a conditionally active anti-AXL antibody or antigen binding antibody fragment that comprises a heavy chain variable region encoded by the nucleotide sequence of SEQ ID NO: 12 and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO: 9.

[0021] In another specific embodiment of the foregoing antibodies and antibody fragments, the present disclosure relates to a conditionally active anti-AXL antibody or antigen binding antibody fragment that comprises an mAbBA3011 antibody encoded by the nucleic acid sequences of SEQ ID NOS. 9 and 12 and having a heavy chain amino acid sequence of SEQ ID NO: 128 and a light chain amino acid sequence of SEQ ID NO: 129.

[0022] In another specific embodiment of the foregoing antibodies and antibody fragments, the present disclosure relates to a pharmaceutical composition comprising AbCAB-(linker-D)n and a pharmaceutically acceptable carrier. In a preferred embodiment, this pharmaceutical composition comprises mAbBA-301 l-(mc-vc-PAB-MMAE)4.

[0023] In each of the foregoing embodiments the linker may be selected from mc-vc-PAB, SPC-BA- 007, SPC-BA-017 or SPC-BA-0019, or the linker is mc-vc-PAB, or the linker is SPC-BA-007, or the linker is SPC-BA-0017, or the linker is SPC-BA-0019.

[0024] In each of the foregoing embodiments, the number of anti-cancer molecules linked to the conditionally active anti-AXL antibody or antibody fragment may vary from 1 to 4, inclusive, or a single anti-cancer molecule may be linked to each conditionally active anti-AXL antibody or antigenbinding antibody fragment, or two anti-cancer molecules may be linked to each conditionally active anti-AXL antibody or antigen binding antibody fragment, or three anti-cancer molecules may be linked to each conditionally active anti-AXL antibody or antibody fragment, or. four anti-cancer molecules may be linked to each conditionally active anti-AXL antibody or antibody fragment.

[0025] In each of the foregoing embodiments, the anti-cancer molecule may be an auristatin, or the anti-cancer molecule may be monomethyl auristatin E (MMAE). In each of the foregoing embodiments, the number of MMAE molecules linked to the conditionally active anti-AXL antibody or antigen binding antibody fragment can vary from 1 to 4, inclusive, a single MMAE molecule may be linked to each conditionally active anti-AXL antibody or antibody fragment, or two MMAE molecules are linked to each conditionally active anti-AXL antibody or antibody fragment, or three MMAE molecules may be linked to each conditionally active anti-AXL antibody or antibodyfragment, or four MMAE molecules are linked to each conditionally active anti-AXL antibody or antibody fragment. In some embodiments, the conditionally active anti-AXL antibody comprises an mAbBA3011 antibody encoded by nucleic acid sequences SEQ ID NOS. 9 and 12 and having a heavy chain amino acid sequence of SEQ ID NO: 128 and a light chain amino acid sequence of SEQ ID NO: 129.

[0026] In each of the foregoing embodiments, the adenoid cystic carcinoma may be a type I adenoid cystic carcinoma, or a type II adenoid cystic carcinoma or a combination of a type I adenoid cystic carcinoma and a type II adenoid cystic carcinoma. In each of the foregoing embodiments the adenoid cystic carcinoma may be a type I adenoid cystic carcinoma. In each of the foregoing embodiments, the adenoid cystic carcinoma may be a type II adenoid cystic carcinoma.

[0027] In each of the preceding embodiments, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier having a pH of 6.0 and comprising 20 mM histidine-HCl, 70 mg / mL sucrose and 0.5 mg / mL polysorbate 80.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1 A-1B show sequence alignments of the heavy chain variable regions and the light chain variable regions, respectively, of anti-AXL antibodies of the present disclosure.

[0029] FIG. 2 shows pH dependent binding profiles of the anti-AXL antibodies of the present disclosure to AXL in KREBS buffer.DEFINITIONS

[0030] In order to facilitate understanding of the examples provided herein, certain frequently occurring terms are defined herein.

[0031] In connection with a measured quantity, the term "about" as used herein refers to the normal variation in that measured quantity that would be expected by a skilled person making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. Unless otherwise indicated, a normal variation is a variation of + / - 10% of the value provided.

[0032] The term “affinity” as used herein refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody andantigen). The affinity of a molecule X for its partner ¥ can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0033] The term “affinity matured” when used in reference to an antibody or antibody fragment refers to an antibody or antibody fragment with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody or antibody fragment which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody or antibody fragment for an antigen.

[0034] The term “alkyl” or “alkylene” as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 20 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also refer to a Ci alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, Ci-Ce alkyl, C1-C7 alkyl, Ci-Cs alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C1-C20 alkyl.

[0035] The term "amino acid" as used herein refers to any organic compound that contains an amino group (-NH2) and a carboxyl group (— COOH); preferably either as free groups or alternatively after condensation as part of peptide bonds. The "twenty naturally encoded polypeptide-forming alphaamino acids" are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0036] The term “agent” as used herein means an element, compound, or molecular entity, including, e.g., a pharmaceutical, therapeutic, or pharmacologic compound. Agents can be natural or synthetic or a combination thereof.

[0037] An “anti-cancer agent” is an agent that exerts a cytotoxic or cytostatic effect on cancer cells either alone or in combination with another agent as part of a treatment regimen. For example, an anti-cancer agent is an agent that can inhibit tumor growth, arrest tumor growth, and / or cause the regression of already existing tumors.

[0038] Cytotoxic effect” means an inhibition of cell proliferation. A “cytotoxic agent” means an agent that has a cytotoxic or cytostatic effect on a cell, thereby depleting or inhibiting the growth of, respectively, cells within a cell population.

[0039] The term “antibody” as used herein refers to intact immunoglobulin molecules. Antibodies or antibody fragments can be used to isolate preparative quantities of an antigen by immunoaffinity chromatography. Various other uses of such antibodies or antibody fragments are to diagnose and / or stage disease (e.g., neoplasia) and for therapeutic application to treat disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like. Chimeric, human-like, humanized or fully human antibodies or antibody fragments are particularly useful for administration to human patients.

[0040] The term “antibody fragment” as used herein refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; and single-chain antibody molecules (e.g. scFv). These antibody fragments, which retain some ability to selectively bind to an antigen (e.g., a polypeptide antigen) of the antibody from which they are derived, can be made using well known methods in the art.

[0041] An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.

[0042] An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab1fragments are obtained per antibody molecule treated in this manner.

[0043] An (Fab')? fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab')2 fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.

[0044] An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains

[0045] The terms “anti-AXL antibody”, “anti-AXL antibody fragment” and “an antibody or antibody fragment that binds to AXL” as used herein refer to an antibody or antibody fragment that is capable of binding AXL with sufficient affinity such that the antibody or antibody fragment is useful as a diagnostic and / or therapeutic agent in targeting AXL. In one embodiment, the extent of binding of an anti-AXL antibody or antibody fragment to an unrelated, non-AXL protein is less than about 10% of the binding of the antibody or antibody fragment to AXL as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody or antibody fragment that binds to AXL has a dissociation constant (Kd) of ^1 x IO"6M (pM), ^100 x 10"9M (nM), ^10 nM, 1 nM, ^0.1 nM, ^0.01 nM, or 5=0.001 nM (e.g. 10XM or less, or from 10 to 10l3M, or from 109M to 10,3M). In certain embodiments, an anti-AXL antibody or antibody fragment binds to an epitope of AXL that is conserved among AXL from different species.

[0046] The term “AXL” as used herein, refers to any native AXL from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed AXL as well as any form of AXL that results from processing in the cell. The term also encompasses naturally occurring variants of AXL, e.g., splice variants or allelic variants. The amino acid sequence of human AXL is well-known in the art and available from public databases such as GenBank.

[0047] The term “AXL activation” as used herein refers to activation, or phosphorylation, of the AXL receptor. Generally, AXL activation results in signal transduction (e.g. that caused by an intracellular kinase domain of an AXL receptor phosphorylating tyrosine residues in AXL or a substrate polypeptide). AXL activation may be mediated by AXL ligand (Gas6) binding to an AXL receptor of interest. Gas6 binding to AXL may activate a kinase domain of AXL and thereby result in phosphorylation of tyrosine residues in the AXL and / or phosphorylation of tyrosine residues in additional substrate polypeptides(s).

[0048] The term “AXL mediated anti-apoptosis” as used herein refers to all AXL-involving processes that prevent human cells, preferably but not limited to human cancer cells, from programmed cell death (apoptosis). In particular, it refers to processes that prevent human cells, preferably but not limited to, human cancer cells from induction of apoptosis through growth factor withdrawal, hypoxia, exposure to chemotherapeutic agents or radiation, or initiation of the Fas / Apo- 1 receptor-mediated signaling, and are stimulated or mediated by non-catalytic or catalytic activities of AXL, preferably including AXL phosphorylation and / or AXL-mediated signal transduction.

[0049] The term "binding" as used herein refers to interaction of the variable region or an Fv of an antibody with an antigen with the interaction depending upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a specific protein structure rather than to proteins generally. As used herein, the term "specifically binding" or "binding specifically" means that an antibody variable region or Fv binds to or associates with more frequently, more rapidly, with greater duration and / or with greater affinity, a particular antigen than with other proteins. For example, an antibody variable region or Fv specifically binds to its antigen with greater affinity, avidity, more readily, and / or with greater duration than it binds to other antigens. For another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with materially greater affinity than it does to related proteins or other cell surface proteins or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans). However, "specifically binding" does not necessarily require exclusive binding or non-detectable binding of another antigen, which instead is meant by the term "selective binding". In one example, "specific binding" of an antibody variable region or Fv (or other binding region) that binds to an antigen, means that the antibody variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100 nM or less, such as 50 nM or less, for example 20 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, 2 nM or less, or 1 nM or less.

[0050] The terms “cell proliferative disorder” and “proliferative disorder” as used herein refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0051] The term “chimeric” antibody as used herein refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0052] The term “class” of an antibody as used herein refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG?, IgG?, IgG4, IgAi, and IgA?. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, 8, y, and p, respectively.

[0053] The terms "conditionally active antibody" and “conditionally active antibody fragment” as used herein refer to an antibody or antibody fragment which is more active at a value of a condition in a tumor microenvironment compared to a different value of the same condition in a non-tumormicroenvironment. As compared to the conditions in the non-tumor microenvironment, the conditions in the tumor microenvironment may include a lower pH, a higher concentration of lactate and / or pyruvate, hypoxia, a lower concentration of glucose, and a slightly higher temperature. For example, in one embodiment a conditionally active antibody or antibody fragment may be virtually inactive at a normal body temperature, but active at a higher temperature that may be encountered in a tumor microenvironment. In yet another embodiment, the conditionally active antibody or antibody fragment may be less active in normal oxygenated blood than in a less oxygenated environment that may exist in a tumor microenvironment. In another embodiment, a conditionally active antibody or antibody fragment may be less active at a normal physiological pH, for example plasma pH, as compared to a greater activity in the more acidic pH of the tumor microenvironment. Generally, the tumor microenvironment has a pH ranging from about pH 5.8 to pH 7.0. In some instances, the tumor microenvironment pH ranges from 5.8 to 6.7, in other instances, the tumor microenvironment pH ranges from about 6.2 to 6.8, while in still other instances the tumor microenvironment pH ranges from about 6.4 to 6.8. There are other conditions in the tumor microenvironment known to a person skilled in the field that may also be selected for use as the condition in the present invention which may trigger the anti-AXL antibodies or antibody fragments to have different activities at different values of that condition.

[0054] The term “constitutive” as used herein, as for example applied to AXL activity, refers to continuous signaling activity of the receptor kinase that is not dependent on the presence of a ligand or other activating molecules. Depending on the nature of the receptor kinase, all of the activity may be constitutive or the activity of the receptor may be further activated by the binding of other molecules (e.g. ligands). Cellular events that lead to activation of receptor kinase are well known among those of ordinary skill in the art. For example, activation may include oligomerization, e.g., dimerization, trimerization, etc., into higher order receptor complexes. Complexes may comprise a single species of protein, i.e., a homomeric complex. Alternatively, complexes may comprise at least two different protein species, i.e., a heteromeric complex. Complex formation may be caused by, for example, overexpression of normal or mutant forms of receptor on the surface of a cell. Complex formation may also be caused by a specific mutation or mutations in a receptor.

[0055] The term “cytostatic agent” as used herein refers to a compound or composition which arrests growth of a cell either in vitro or in vivo. Thus, a cytostatic agent may be one which significantly reduces the percentage of cells in S phase. Further examples of cytostatic agents include agents that block cell cycle progression by inducing G0 / G1 arrest or M-phase arrest. The humanized anti-Her2antibody trastuzumab (HERCEPTIN®) is an example of a cytostatic agent that induces G0 / G1 arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Certain agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (W.B Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semi synthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

[0056] The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to radioactive isotopes (e.g., At211, 1131, 1125, Y90, Re186, Re188, Sm1’3, Bi212, P32, Pb212and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and the various antitumor or anti cancer agents disclosed herein.

[0057] The term “diabodies” as used herein refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a lightchain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0058] The term “effective amount” of an agent as used herein, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0059] The term “Fc region” as used herein is used to define a C -terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0060] The terms “framework”, “framework region” or “FR” as used herein refer to variable domain residues other than hypervariable region (HVR or Hl -3 in the heavy chain and LI -3 in the light chain) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FRS, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0061] The terms “full length antibody,” “intact antibody,” or “whole antibody” refer to an antibody which comprises an antigen-binding variable region (VH or VL) as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e g. human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domain of their heavy chains, full length antibodies can be assigned to different “classes”. There are five major classes of full length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0062] The term “human antibody” as used herein is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non- human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues.

[0063] The term “humanized” antibody as used herein refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0064] The terms “hypervariable region” or “HVR” as used herein refer to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol., vol. 196, pp. 901-917 1987) Exemplary CDRs (CDR- Ll, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of Hl, 50-65 of H2, and 95-102 of H3 (Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991). With the exception of CDR1 in the VH, CDRS generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact the antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-Ll, a-CDR-L2, a- CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of Hl, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008).

[0065] The term “immunoconjugate” as used herein is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0066] The term “individual” or “subject” as used herein refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0067] “Adverse Event” (AE) (also referred to as an adverse experience) means any unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease temporally associated with the use of a drug and does not imply any judgment about causality. An AE can arise with any use of a drug (e.g., off-label use or use in combination with another drug) and with any route of administration, formulation, or dose, including an overdose. An AE is considered “serious” if it results in any of the following outcomes:• death (excluding death due to underlying disease),• is life-threatening,• requires inpatient hospitalization or prolongation of existing hospitalization,• a persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions,• a congenital anomaly / birth defect, and• is an important medical event - Important medical events that may not result in death, be lifethreatening, or require hospitalization may be considered serious when, based upon appropriate medical judgment, may jeopardize the patient and / or may require medical or surgical intervention to prevent 1 of the outcomes listed in this definition.

[0068] The terms “inhibit” or “inhibition of’ as used herein mean to reduce by a measurable amount, or to prevent entirely.

[0069] The term “inhibiting cell growth or proliferation” as used herein means decreasing a cell's growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0070] The term “isolated” antibody as used herein refers to an antibody which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), or capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) means. For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B, vol. 848, pp. 79-87, 2007.

[0071] The term “metastasis” as used herein refers to processes that support cancer cells to disperse from a primary tumor, penetrate into lymphatic and / or blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasis) in normal tissues elsewhere in the body. In particular, “metastasis” refers to cellular events of tumor cells such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors, that underlie metastasis.

[0072] The term "microenvironment" as used herein means any portion or region of a tissue or body that has constant or temporal, physical or chemical differences from other regions of the tissue or regions of the body. For tumors, the term “tumor microenvironment” as used herein refers to the environment in which a tumor exists, which is the non-cellular area within the tumor and the area directly outside the tumorous tissue but does not pertain to the intracellular compartment of the cancer cell itself. The tumor and the tumor microenvironment are closely related and interact constantly. A tumor can change its microenvironment, and the microenvironment can affect how a tumor grows andspreads. Typically, the tumor microenvironment has a low pH in the range of 5.8 to 7.0, while in some instances the pH range is 5.8 to 6.7 while in other instances the pH range is 6.2 to 6.8, while in still other instances the pH range is 6.4-6.8. On the other hand, a normal physiological pH is typically in the range of 7.2-7.8. The tumor microenvironment is also known to have a lower concentration of glucose and other nutrients, but a higher concentration of lactic acid, in comparison with blood plasma. Furthermore, the tumor microenvironment can have a temperature that is 0.3 °C to 1 °C higher than the normal physiological temperature. The tumor microenvironment has been discussed in Gillies et al., “MRI of the Tumor Microenvironment,” Journal of Magnetic Resonance Imaging, vol. 16, pp.430- 450, 2002. The term “non-tumor microenvironment” refers to a microenvironment at a site other than a tumor.

[0073] The term “percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence as used herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN- 2 program is publicly available from Genentech, Inc., South San Francisco, Calif, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0074] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprisesa certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:100 * (X / Y) where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0075] The term “pharmaceutical formulation” as used herein refers to a preparation which is in a form that permits the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation is to be administered.

[0076] The term “pharmaceutically acceptable carrier” as used herein refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, an excipient, a stabilizer, a preservative or any combination thereof.

[0077] The term “recombinant antibody” as used herein refers to an antibody (e.g. a chimeric, humanized, or human antibody or antigen-binding fragment thereof) that is expressed by a recombinant host cell comprising nucleic acids encoding the antibody. Examples of “host cells” for producing recombinant antibodies include: (1) mammalian cells, for example, Chinese Hamster Ovary (CHO) cells, COS cells, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK) cells, Hela cells and Vero cells; (2) insect cells, for example, sf9, sf21 and Tn5; (3) plant cells, for example plants belonging to the genus Nicotiana (e.g. Nicotiana tabacuniy, (4) yeast cells, for example, those belonging to the genus Saccharomyces (e.g. Saccharomyces cerevisiae) or the genus Aspergillus (e g. Aspergillus niger), (5) bacterial cells, for example Escherichia, colt cells or Bacillus subtilis cells, etc.

[0078] The term “therapeutically effective amount” of the antibody or antibody fragment of the invention means a sufficient amount of the antibody or antibody fragment to treat a disease or illness, at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the antibodies or antibody fragments and compositions of the presentdisclosure may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated, the severity of the disorder; the activity of the specific antibody or antibody fragment employed; the specific composition employed, the age, body weight, general health, sex and / or diet of the patient; the time of administration, the route of administration, the rate of excretion of the specific antibody or antibody fragment employed; the duration of the treatment, drugs used in combination or coincidental with the specific antibody employed, and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0079] The term “single chain Fv” (“scFv”) as used herein refers to a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. “dsFv” is a VH:VL heterodimer stabilized by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs with a peptide linker, such as divalent sc(Fv)2.

[0080] The term “treatment,” “treat,” or “treating” as used herein refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies or antibody fragments of the invention are used to delay development of a disease or to slow the progression of a disease. In particular embodiments, antibodies or antibody fragments of the invention are used to prevent occurrence or recurrence of tumor proliferation, alleviate symptoms related to tumor progression or regression, diminish any direct or indirect pathological consequences related to cancer, prevent tumor metastasis, enhance tumor regression, decrease or inhibit tumor progression, and induce remission or improved prognosis.

[0081] The term “tumor” as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

[0082] The term “variable region” or “variable domain” as used herein refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies or antibody fragments that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; and Clarkson et al., Nature, vol. 352, pp. 624-628, 1991.DETAILED DESCRIPTION

[0083] For illustrative purposes, the principles of the present invention are described by referencing various exemplary embodiments. Although certain embodiments of the invention are specifically described herein, one of ordinary skill in the art will readily recognize that the same principles are equally applicable to, and can be employed in, other systems and methods. Before explaining the disclosed embodiments of the present invention in detail, it is to be understood that the invention is not limited in its application to the details of any particular embodiment shown. Additionally, the terminology used herein is for the purpose of description and not for limitation. Furthermore, although certain methods are described with reference to steps that are presented herein in a certain order, in many instances, these steps can be performed in any order as may be appreciated by one skilled in the art; the novel method is therefore not limited to the particular arrangement of steps disclosed herein.

[0084] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Furthermore, the terms “a” (or a “an”), “one or more”, and “at least one” can be used interchangeably herein. The terms “comprising”, “including”, “having” and “constructed from” can also be used interchangeably.

[0085] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, ratio, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about,” whether or not the term “about” is present. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be constmed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0086] It is to be understood that each component, compound, substituent, or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more of each and every other component, compound, substituent, or parameter disclosed herein.

[0087] It is also to be understood that each amount / value or range of amounts / values for each component, compound, substituent, or parameter disclosed herein is to be interpreted as also being disclosed in combination with each amount / value or range of amounts / values disclosed for any other component(s), compounds(s), substituent(s), or parameter(s) disclosed herein and that any combination of amounts / values or ranges of amounts / values for two or more component(s), compounds(s), substituent(s), or parameters disclosed herein are thus also disclosed in combination with each other for the purposes of this description.

[0088] It is further understood that each lower limit of each range disclosed herein is to be interpreted as disclosed in combination with each upper limit of each range disclosed herein for the same component, compounds, substituent, or parameter. Thus, a disclosure of two ranges is to be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. A disclosure of three ranges is to be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, etc. Furthermore, specific amounts / values of a component, compound, substituent, or parameter disclosed in the description or an example is to be interpreted as a disclosure of either a lower or an upper limit of a range and thus can be combined with any other lower or upper limit of a range or specific amount / value for the same component, compound, substituent, or parameter disclosed elsewhere in the application to form a range for that component, compound, substituent, or parameter.

[0089] Provided herein is a method of treating adenoid cystic carcinoma, comprising administering to a subject having adenoid cystic carcinoma a pharmaceutical composition comprising AbCAB-(linker- D)nand a pharmaceutically acceptable carrier at a dose of 1.8 mg / kg once every two weeks, wherein AbCAB is a conditionally active anti-AXL antibody or antibody fragment, linker is a cleavable linker,D is an anti-cancer agent, and n is an integer from 1 to 4. Also provided herein is a pharmaceutical composition for administration to a subject having adenoid cystic carcinoma at a dose of 1.8 mg / kg once every two weeks, comprising AbCAB-(linker-D)nand a pharmaceutically acceptable carrier, wherein AbCAB is a conditionally active anti-AXL antibody or antibody fragment, linker is a cleavable linker, D is an anti-cancer agent, and n is an integer from 1 to 4.Conditionally Active Anti-AXL Antibodies or Antibody Fragments

[0090] Conditionally active anti-AXL antibodies or antibody fragments of the present disclosure include six complementarity determining regions Hl, H2, H3, LI, L2 and L3, wherein: the Hl sequence is X1GX2X3MX4 (SEQ ID NO: 1) (Xi, X2, X3 and X4each independently represent an amino acid): wherein:Xi is T or A or W,X2 is H or A,X3 is T or I, andX4is N or I; the H2 sequence is LIKXsSNGGTXeYNQKFKG (SEQ ID NO: 2) (X5and X6each independently represent an amino acid): wherein:X5 is P or N, andXe is S or I or T; and the H3 sequence is GX7X8X9X10X11X12X13X14DYX15X16 (SEQ ID NO: 3) (X7, X8, X9, Xto, Xu, Xi2, X13, X14, X15, and Xi6 each independently represent an amino acid): wherein: X? is H or D or E or P or R or W, Xg is Y orN,X9 is E or A or D or F or G or H or I or L or M or N or R or V or Y,X10 is S or D or M or N or Q,Xu is Y or C or E or P,X12 is F or E or N or S or T or V,X13 is A or D or G or L or Y,X14 is M or E or F,X15 is W or A or D or H or L or N or P or R or T, andXi6 is G or H, the LI sequence is KASQDX17X18SX19VX20 (SEQ ID NO: 4) (Xi7, Xis, X19, and X20each independently represent an amino acid): wherein:X17 is V or D or G or N or W,Xi8 is S or V,X19 is A or L or M, andX20 is A or D or N or Q; the L2 sequence is X21X22X23TRX24T (SEQ ID NO: 5) (X21, X22, X23, and X24, each independently represent an amino acid): wherein:X21 is W or F,X22 is A or I or N or P or Q,X23 is S or DX24 is H or D; and the L3 sequence is QEX25X26SX27X28X29X30 (SEQ ID NO: 6) (X25, X26, X27, X28, X29, andX30, each independently represent an amino acid): wherein:X25 is H or C or F or I or L or Q or S or T or V or Y,X26 is F or C or D or E or G or N or S,X27 is T or C or P,X28 is P or A or C or D or E or H or K or S or T or V or W,X29 is L or G or R, andX30 is T or I or R, and the anti-AXL antibody or antibody fragment excludes an antibody or antibody fragment having the following six CDRs:Hl - TGHTMN,H2 = LIKPSNGGTSYNQKFKG,H3 GHYESYFAMDYWG,LI = KASQDVSSAVA,L2 = WASTRHT, andL3 = QEHFSTPLT.

[0091] In one aspect is provided a conditionally active anti-AXL antibody or antibody fragment which has up to three substitutions in CDRs Hl, H2 and H3, relative to the HC-WT in Fig. 1A and up to five substitutions in CDRs LI, L2 and L3, relative to the LC-WT in Fig. IB. This includes conditionally active anti-AXL antibodies or antibody fragments with one substitution in CDRs Hl, H2 and H3, relative to the HC-WT in Fig. 1A, conditionally active anti-AXL antibodies or antibody fragments with one substitution in CDRs LI, L2 and L3, relative to the LC-WT in Fig. IB, and conditionallyactive anti-AXL antibodies or antibody fragments with one substitution in CDRs Hl, H2 and H3, and one substitution in CDRs LI, L2 and L3, relative to the HC-WT in Fig. 1A and the LC-WT in Fig IB. The possible combinations of CDRs Hl, H2 and H3 are shown in Fig. 1A and the possible combinations of CDRs LI, L2 and L3 are shown in Fig. IB In one aspect, the combination of the CDRs in the HC-WT in Fig. 1 A with the CDRs of the LC-WT in Fig. IB is excluded from the possible combinations.

[0092] The alignment of the heavy chain variable regions is shown in FIG 1A, where the complementarity determining regions Hl, H2, and H3 are boxed.

[0093] The alignment of the light chain variable regions is shown in FIG. IB, where the complementarity determining regions LI, L2, and L3 are boxed.

[0094] The conditionally active anti-AXL antibodies and antibody fragments disclosed herein were obtained from a wild-type (WT) antibody whose heavy chain and light chain variable regions are disclosed in Figs. 1 A and IB using the method disclosed in U.S. Patent No. 8,709,755.

[0095] The DNA encoding the wild-type antibody was evolved to generate a mutant antibody library using Comprehensive Positional Evolution (CPE), whereby each position in the template antibody is randomized one at a time. Each mutant antibody in the library has only one single point mutation. The mutant antibodies in the library were generated by simultaneously screening for selective binding affinity to AXE at pH 6.0 over pH 7.4 by ELISA. Two mutant antibody dilutions were used: 1:3 and 1:9 dilutions. The mutant antibodies that have at least a 1.5 ratio of binding affinity at pH 6.0 to the binding affinity at pH 7.4 under either the 1 :3 or the 1:9 dilution are selected as conditionally active antibodies, with the single point mutations indicated in each of the heavy chain and light chain variable regions (see Tables 1 and 2). Thank you for uploading the image. I have performed OCR on the provided table, which appears to show data from an Affinity ELISA CPE (Cytopathic Effect) mutant assay, with ratio measurements at different pH levels (1:3, pH 6.0 / 7.4 and 1:9, pH 6.0 / 7.4). Below is the extracted and organized data:Table 1: Conditionally active anti-Axl antibody heavy chain variable regionsTable 2: Conditionally active anti-Axl antibody light chain variable region

[0096] The method identified the heavy chain variable regions as presented in FIG. 1A and the light chain variable regions as presented in FIG IB. Some heavy chain variable regions are encoded by DNA sequences with SEQ ID NOS: 11-13. Some light chain variable regions are encoded by DNA sequences with SEQ ID NOS: 7-10. These heavy and light chain variable regions can specifically bind to AXL. Antibodies comprising one of these heavy and light chain variable regions have been found to have a higher binding affinity to AXL at a pH found in the tumor microenvironment than at a pH in a non-tumor microenvironment.

[0097] In one embodiment, the present disclosure also includes variants of the heavy and light chain variable regions presented in FIGS. 1A-1B and encoded by DNA sequences with SEQ ID NOS: 9-13 that can specifically bind to AXL In order to derive these variants, it was determined that the complementarity determining regions (CDRs) of the heavy chain variable regions (H1-H3) and the CDRs of the light chain variable regions (L1-L3) should remain intact.

[0098] One specific embodiment provides a conditionally active anti-AXL antibody or antibody fragment containing six complementarity determining regions Hl, H2, H3, LI, L2 and L3 in which Xi is W, X2is A, X3is T, X4is N, X5is P, X6is S, X7is H, X8is Y, X9is E, Xi0is S, Xu is Y, Xi2is E, Xi3 is A, X14 is M, Xis is W, Xi6 is G, Xi? is V, Xis is V, X19 is A, X20 is A, X21 is W, X22 is Q, X23 is D, X24 is H, X25 is H, X26 is F, X27 is P, X28 is P, X29 is L and X30 is T.

[0099] Another specific embodiment provides a conditionally active anti-AXL antibody or antibody fragment containing six complementarity determining regions Hl, H2, H3, LI, L2 and L3 in which Hl is WGATMN, H2 is LIKPSNGGTSYNQKFKG, H3 is GHYESYEAMDYWG, LI is KASQDVVSAVA, L2 is WQDTRHT and L3 is QEHFSPPLT.

[0100] In deriving these variants, one is guided by the process as described herein. The variants of these heavy and light chain variable regions may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the heavy and light chain variable regions, or by peptide synthesis. Such modifications include, for example, deletion(s) from, and / or insertion(s) into and / or substitution(s) of residues within the amino acid sequences of the antibody or antibody fragment. Any combination of deletion(s), insertion(s), and substitution(s) can be made to arrive at the final construct, provided that the final construct possesses at least one of the desired characteristics, e.g., antigenbinding.Fc Region Variants

[0101] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the conditionally active anti-AXL antibodies or antibody fragments provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

[0102] Certain embodiments contemplate an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important, yet certain effector functions (such as ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 5 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. , vol. 9, pp 457-492, 1991 Non-limitingexamples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see also, e.g. Hellstrom et al. Proc. Nat'l Acad. Sci. USA, vol. 83, pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, vol. 82, pp. 1499-1502, 1985; U.S. Pat. No. 5,821,337 and Bruggemann et al., J. Exp. Med, vol. 166, pp. 1351-1361, 1987. Alternatively, nonradioactive assay methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.; and CytoTox 96® nonradioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISAin WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, vol. 202, pp. 163-171, 1996; Cragg, M. S. et al., Blood, vol. 101, pp. 1045- 1052, 2003; and Cragg, M. S, and M J. Glennie, Blood, vol. 103, pp. 2738-2743, 2004). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B et al., Int'l Immunol., vol. 18, pp. 1759-1769, 2006).

[0103] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 with alanine (U.S. Pat. No. 7,332,581).

[0104] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0105] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6, 194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., vol. 164, pp. 4178-4184, 2000.

[0106] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol, vol. 117, pp. 587-593, 1976 and Kim et al., J. Immunol, vol. 24, p. 249, 1994), are described inUS2005 / 0014934. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 and 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.Immunoconj ugates

[0107] The present disclosure also provides immunoconjugates comprising an anti-AXL antibody as described herein conjugated to one or more anti-cancer agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radioactive isotopes.

[0108] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more anti-cancer agents including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0425 235 Bl); an auristatin such as monomethylauristatin drug moieties DE and DE (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res., vol. 53, pp. 3336-3342, 1993; and Lode et al., Cancer Res., vol. 58, pp. 2925-2928, 1998); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem., vol. 13, pp. 477-523, 2006; Jeffrey et al., Bloor ganic & Med. Chem. Letters, vol. 16, pp. 358-362, 2006; Torgov et $)., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc. Natl. Acad. Set. USA, vol. 97, pp. 829-834, 2000, Dubowchik et al., Bioorg. & Med. Chem. Letters, vol. 12, vol. 1529- 1532, 2002; King et al., J. Med. Chem., vol. 45, pp. 4336-4343, 2002; and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC 1065. In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa)' , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP- S), momordica charantia inhibitor, curcin, crotin, sapaonana officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0109] In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At211, 1131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212and radioactive isotopes of Lu.

[0110] Conjugates of an antibody and anti-cancer agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3 -(2 -pyridyldithio) propionate (SPDP), succinimidyl- 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, vol. 238, starting at p. 1098, 1987. Carbon- 14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to the antibody. See WO94 / 11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing linker (Chari et ah, Cancer Res., vol. 52, pp. 127-131, 1992; U.S. Pat. No. 5,208,020) may be used.

[0111] The immunoconjugates herein include, but are not limited to, conjugates prepared with crosslinker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo- MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

[0112] An exemplary embodiment of an ADC comprises an antibody (Ab) which targets a tumor cell, an anti-cancer agent (D), and a linker moiety (L) that attaches the Ab to D. In some embodiments, the antibody is attached to the linker moiety (L) through one or more amino acid residues, such as lysine and / or cysteine.

[0113] An exemplary ADC has the formula AbCAB-(L-D)n, where n is 1 to about 20, inclusive. In certain embodiments, n is 1-10, while in other embodiments, n is 1-5. In still other embodiments, n is 1-4. In further embodiments, n is 1-3. In additional embodiments, is 1 or 2. In still further embodiments n is 1. In some embodiments, the number of drug moi eties that can be conjugated to an antibody islimited by the number of free cysteine residues. In some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. Exemplary ADC disclosed herein, include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon et al., Methods inEnzym., vol. 502, pp. 123-138, 2012). In some embodiments, one or more free cysteine residues are already present in an antibody, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody to a drug. In some embodiments, an antibody is exposed to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues.Exemplary Linkers

[0114] A “Linker” (L) is a bifunctional or multifunctional moiety that can be used to link one or more moieties such as anti-cancer agents (D) to an antibody (Ab) to form an immunoconjugate such as provided herein. In some embodiments, ADCs can be prepared using a Linker having reactive functionalities for covalently attaching to the anti-cancer agent and to the antibody. For example, in some embodiments, a cysteine thiol of an antibody (Ab) can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.

[0115] In one aspect, the linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4- nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., the conjugation method at page 766 of Klussman, et al, Bioconjugate Chemistry, vol. 15, pp. 765-773, 2004.

[0116] In some embodiments, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemplary electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Nonlimiting exemplary such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0117] A linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-l carboxylate (“MCC”). Various linker components are known in the art and may be employed in the present invention.

[0118] A linker may be a “cleavable linker,” facilitating release of a drug. Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, and disulfide-containing linkers (Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992; U.S. Pat. No. 5,208,020).

[0119] In certain embodiments, a linker has the formula — Aa— Www —— Y Yy— , wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W is an “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacer unit”, and y is 0, 1, or 2. An ADC comprising this linker has the formula : Ab-(Aa— -1Ww— Yy-D)n, wherein Ab, D, and n are as defined herein. Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298.

[0120] In some embodiments, a linker component comprises a “stretcher unit” (A) that links an antibody to another linker component or to a drug moiety. Nonlimiting exemplary stretcher units are shown below (wherein the wavy line indicates sites of covalent attachment to an antibody, drug, or additional linker components):

[0121] In some embodiments, a linker component comprises an “amino acid unit” (W). In some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al., Nat. Biotechnol., vol. 21, pp. 778-784, 2003). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alaninephenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe- homolys); and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). An amino acid unit may comprise amino acid residues that occur naturally and / or minor amino acids and / or non- naturally occurring amino acid analogs, such as citrulline amino acid units, can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[0122] Peptide-type linkers can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method (e.g., E. Schroder and K. Ltibke (1965) “The Peptides”, volume 1, pp 76-136, Academic Press).

[0123] In some embodiments, a linker component comprises a “spacer unit” (Y) that links the antibody to a drug moiety, either directly or through a stretcher unit and / or an amino acid unit. A spacer unit may be “self-immolative” or a “non-self-immolative.” A “non-self-immolative” spacer unit is one in which part or all of the spacer unit remains bound to the drug moiety upon cleavage of the ADC. Examples of non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. In some embodiments, enzymatic cleavage of an ADC containing aglycine-glycine spacer unit by a tumor-cell associated protease results in release of a glycine-glycine- drug moiety from the remainder of the ADC. In some such embodiments, the glycine-glycine-drug moiety is subjected to a hydrolysis step in the tumor cell, thus cleaving the glycine-glycine spacer unit from the drug moiety.

[0124] A“self-immolative” spacer unit allows for release of the drug moiety. In certain embodiments, a spacer unit of a linker comprises a p-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol is attached to an amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the benzyl alcohol and the drug (Hamann et al. Expert Opin. Ther Patents, vol. 15, pp. 1087-1103, 2005). In some embodiments, the spacer unit comprises p- aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising a self-immolative linker has the structure:wherein Q is — Ci-Cs alkyl, — O — (Ci-Cg alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; X may be one or more additional spacer units or may be absent; and p ranges from 1 to about 20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4. Nonlimiting exemplary X spacer units include:wherein Ri and R2 are independently selected from H and Ci-Ce alkyl groups. In some embodiments, Ri and R2 are each — CH3.

[0125] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al., Bioorg. Med. Chem. Lett., vol. 9, p. 2237-, 1999) and ortho- or para-aminobenzylacetals. In some embodiments, spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, vol. 2, pp. 223-, 1995), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., vol. 94, p. 5815-, 1972) and 2- aminophenylpropionic acid amides (Amsberry et al, J. Org. Chem., vol. 55, p. 5867, 1990). Linkage of a drug to the a-carbon of a glycine residue is another example of a self-immolative spacer that may be useful in ADCs (Kingsbury et al., J. Med. Chem., vol. 27, p.1447, 1984).

[0126] In some embodiments, a linker is substituted with groups that modulate solubility and / or reactivity. As a nonlimiting example, a charged substituent such as sulfonate (-SO3 ) or ammonium may increase water solubility of the linker reagent and facilitate the coupling reaction of the linker reagent with the antibody and / or the drug moiety, or facilitate the coupling reaction of Ab-L (antibodylinker intermediate) with D, or D-L (drug-linker intermediate) with Ab, depending on the synthetic route employed to prepare the ADC. In some embodiments, a portion of the linker is coupled to the antibody and a portion of the linker is coupled to the drug, and then the Ab-(linker portion) is coupled to drug-(linker portion) to form the ADC.

[0127] The ADCs provided herein include, but are not limited to, ADCs prepared with the following linker reagents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(P-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS), N-(s-maleimidocaproyloxy) succinimide ester (EMCS), N-[y- maleimidobutyryloxy] succinimide ester (GMBS), 1,6-hexane-bis-vinyl sulfone (HBVS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxy-(6-amidocaproate) (LC-SMCC), m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), and including bis-maleimide reagents: dithiobismaleimidoethane (DTME), 1,4-bismaleimidobutane (BMB), 1,4 bismaleimidyl-2,3- dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)2 (shown below), and BM(PEG)s (shown below); bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene). In some embodiments, bis-maleimide reagents allow the attachment of the thiol group of a cysteine in the antibody to a thiol-containing drug moiety, linker, or linker-drug intermediate. Other functional groups that are reactive with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

[0128] Certain useful linker reagents can be obtained from various commercial sources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), Molecular Biosciences Inc. (Boulder, Colo.), or synthesized in accordance with procedures described in the art; for example, in Toki et al., J. Org. Chem., vol. 67, pp. 1866-1872, 2002; Dubowchik, et al., Tetrahedron Letters, vol. 38, pp. 5257-60, 1997; Walker, J. Org. Chem., vol. 60, pp. 5352-5355, 1995; Frisch et al., Bioconjugate Chem., vol. 7, pp. 180-186, 1995; U.S. Pat. No. 6,214,345; WO 02 / 088172; US2003130189; US 2003 / 096743; WO 03 / 026577; WO 03 / 043583; and WO 04 / 032828.

[0129] Nonlimiting exemplary linkers are shown below in the context of an ADC disclosed herein are:wherein Ri and Rz are independently selected from H and Ci-Cs alkyl. In some embodiments, Ri and Rz are each -CHy.wherein n is 0 to 12. In some embodiments, n is 2 to 10. In some embodiments, n is 4 to 8.

[0130] Further nonlimiting exemplary ADCs include the structures:each R is independently H or Ci-Ce alkyl; and n is 1 to 12.

[0131] Further non-limiting examples of linker for use in the present disclosure include those having a formula represented bywhere Ri is a nucleophilic functional group capable of coupling to an antibody selected from the group consisting of a C1-C10 alkyl halide, a C1-C10 alkyl alcohol, a phenol, a C1-C10 alkyl thiol, C1-C10 alkyl carboxylic acid, and a C1-C10 alkyl azide, R2 is a substituted or unsubstituted C1-C20 alkylene, or a substituted or unsubstituted (PEG)n moiety represented bywhere n is 1 to 20, andR? is an azide or an acetylene group.

[0132] In an embodiment of the above linker, Ri can be Ci-Ce alkyl halide with a halide selected from the group consisting of Br, I, Cl, and F, a C1-C2 alkyl alcohol, a 4-phenol, or a C1-C2 alkyl thiol.

[0133] In the embodiment of the above linker, Ri can be -CFb-Br.

[0134] In any one of the embodiments of the above linker, R2 can be an unsubstituted C1-C20 alkylene.

[0135] In the above embodiment of the linker, R2 can be an unsubstituted C2-C5 alkylene.

[0136] In any one of the embodiments of the above linker, R2 can be an unsubstituted (PEG)n where n is 8.

[0137] In any one of the embodiments of the above linker, R? can be an azide.

[0138] In any one of the above embodiments, the linker can have a structure represented bywhere p can be 1 to 20, preferably p can be 2 to 5.

[0139] In the above embodiment, the linker can have a structure represented by

[0140] In another of the above embodiments, the linker can have a structure represented by

[0141] In another of the above embodiments, the linker can have a structure represented bywhere n is 8.

[0142] The linker may comprise a substituted or unsubstituted Ci-C2o-slkyl, or a substituted or unsubstituted (PEG)n moiety represented bywhere n is 1-20.

[0143] The “linker” of the present disclosure may be a bifunctional or multifunctional component or moiety that can be used to link one or more drug (D) moieties (payload) to a polypeptide such as an antibody (Ab) to form a drug conjugate, an immunoconjugate, or an antibody-drug conjugate (ADC). Linkers of the present disclosure play a significant role in the stability of drug conjugates and ADCs, and provide a targeted payload release (release of the drug moiety or drug moieties) profile important for the efficacy of ADC drugs. The linker of the present disclosure is stable in the circulatory system, and reduces systemic off-target toxicity by releasing cytotoxic payloads specifically to a target, such as tumor.

[0144] It is desirable to generate a linker of the present disclosure capable of covalently coupling one or more drug moieties to an antibody, or an antibody fragment thereof.

[0145] Specifically, the linker is capable of covalently coupling to an antibody (Ab) or antibody fragment thereof to form ADCs without the defect of releasing payloads nonspecifically in order to optimize the therapeutic windows of ADCs. The linker of the present disclosure may be covalently coupled directly or indirectly to a drug moiety as described herein.

[0146] As described herein, the linker of the present disclosure comprises an antibody coupling group or a nucleophilic functional group capable of coupling to an antibody at one end and a reactive group at the other end that is complementary to an acetylene or azide group of a tether group (“X”) as described herein. Preferably, the reactive group is an azide or acetylene at R? that may react with the acetylene or azide group attached to Rs of the tether group by acetylene-azide cycloaddition to provide a conjugate useful for conjugating to an antibody or antibody fragment of the present disclosure. This cycloaddition reaction between an acetylene moiety and an azide moiety is an example of a “click reaction” or “click chemistry,” the technique of which has been described by Kolb H.C. and K.B. Sharpless, Drug Discov. Today, 8: 1128-37 (2003), the disclosure of which is incorporated herein inits entirety. The click reaction or click chemistry is chemosei ective and may complement other conjugation chemistries such as the thiol-maleimide reaction.

[0147] The antibody coupling group or nucleophilic functional group capable of coupling to an antibody may include, but is not limited to, a C1-C10 alkyl halide, a C1-C10 alkyl alcohol, a phenol, a C1-C10 alkyl thiol, a C1-C10 alkyl carboxylic acid, and a C1-C10 alkyl azide. Preferably, the antibody coupling group or nucleophilic functional group capable of coupling to an antibody is a Ci-Ce alkyl halide, in which the halide is selected from the group consisting of Br, I, Cl, and F, a C1-C2 alkyl alcohol, a 4-phenol, and a C1-C2 alkyl thiol.Exemplary Drug Conjugates

[0148] A tether group (“X”) as described herein may comprise an acetylene or azide group useful for conjugating with a linker of the present disclosure.

[0149] In some instances, and as herein and applicable throughout the present disclosure, a substituent of a substituted alkyl, a substituted aryl, a substituted heteroaryl, a substituted cycloalkyl, or a substituted heterocyclyl, may be selected from the group consisting of a substituted or unsubstituted C1-C20 alkyl, a substituted or unsubstituted C6-C25 aryl, a substituted or unsubstituted C2-C25 heteroaryl, a substituted or unsubstituted C3-C25 cycloalkyl, a substituted C2-C25 heterocyclyl, and / or a PEGn where n is 1-25.

[0150] As described herein and applicable throughout the present disclosure, the substituted or unsubstituted C1-C20 alkyl may be straight chain or branched. The alkyl group includes from 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. For example, the substituted or unsubstituted C1-C20 alkyl may be a substituted or unsubstituted alkylene, such as a substituted or unsubstituted C1-C10 alkylene, or a substituted or unsubstituted Ci-Cs alkylene, a substituted or unsubstituted Ci-Ce alkylene, a substituted or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C1-C4 alkylene, a substituted or unsubstituted C1-C3 alkylene, or a substituted or unsubstituted C1-C2 alkylene. In some instances, the substituted alkyl may include a substituent such as a cleavable moiety, e.g., an enzymatically cleavable moiety like a glycoside or glycoside derivative.

[0151] As described herein and applicable throughout the present disclosure, the substituted or unsubstituted aryl may include a phenyl group. In some instances, the substituted aryl is a substituted phenyl, where the substituent includes a cleavable moiety such as an enzymatically cleavable moiety like a glycoside or glycoside derivative.

[0152] Likewise, in some instances, the substituent on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl may include a cleavable moiety such as an enzymatically cleavable moiety like a glycoside or glycoside derivative.

[0153] In one particular embodiment, the tether group (“X”) is represented by:where Ra can be a C1-C20 alkylene; and R4 can be a triazole ring having a C=C double bond in the ring. The triazole ring of R4 may be formed in a click reaction between an azide group and an acetylene group.

[0154] The present disclosure includes a conjugate of Formula I,wherein:L is represented by:where Ri is a nucleophilic functional group capable of coupling to an antibody selected from the group consisting of a C1-C10 alkyl halide, a C1-C10 alkyl alcohol, a phenol, a C1-C10 alkyl thiol, and a C1-C10 alkyl azide, R2 is a substituted or unsubstituted C1-C20 alkylene, or a substituted or unsubstituted (PEG)nmoiety represented bywhere n can be 1 to 20;X is a tether group, which, in some embodiments, is represented by:where R3 is a C1-C20 alkylene; R4 is a triazole ring having a C=C double bond in the ring;Y is a glycoside or glycoside derivative;D is a drug; and the wavy lines indicate a site of covalent attachment.

[0155] In an embodiment of the above conjugate, Ri is a Ci-Cs alkyl halide with a halide selected from the group consisting of Br, I, Cl, and F, a C1-C2 alkyl alcohol, a 4-phenol, or a C1-C2 alkyl thiol.

[0156] In any one of the embodiments of the above conjugate, Ri is -CFE-Br.

[0157] In any one of the embodiments of the above conjugate, R2 is an unsubstituted C1-C20 alkylene.

[0158] In a specific embodiment of the above conjugate, R2 is an unsubstituted C2-C5 alkylene.

[0159] Alternately, in any one of the embodiments of the above conjugate, R2 is an unsubstituted(PEG)nwhere n is 8.

[0160] In any one of the embodiments of the above conjugate, R3 is -CH2-.

[0161] In any one of the embodiments of the above conjugate, the glycoside Y is selected from the following structures:

[0162] In any one of the embodiments of the above conjugate, the glycoside or glycoside derivativeV is a glucuronide.

[0163] In any one of the embodiments of the above conjugate, the triazole ring of R.4 is formed with an azide group bonded to L which is represented by:where p is an integer of from 2 to 5.

[0164] Alternately, in any one of the embodiments of the above conjugate, the triazole ring is formed with an azide group bonded to L which is represented by:where n is 8.

[0165] In any one of the embodiments of the above conjugate, when R? is a substituted or unsubstituted (PEG)n, Y is not a galactoside.

[0166] In any one of the embodiments of the above conjugate, the drug D is selected from the group consisting of an auristatin, a dolastatin, a maytansinoid, a calicheamicin, a pyrrolobenzodiazepine, an anthracycline, a ribonuclease, and a DNA endonuclease.

[0167] In any one of the embodiments of the above conjugate, the drug D is an auristatin selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0168] Examples of conjugates of the present disclosure include, but are not limited to, the structures shown below.

[0169] In any one of the embodiments of the above conjugate, the conjugate has a structure represented by MC-val-cit-PAB

[0171] To a solution of (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (100 mg, 0.252 mmol, 1.0 eq.) in dry MeCN (2.5 mL, 0.1 m) was added 4-hydroxy-3 -nitrobenzaldehyde (63 mg, 0.38 mmol, 1.5 eq ) and silver oxide (87 mg, 0.38 mmol, 1.5 eq.) at room temperature under a nitrogen atmosphere. The resulting reaction mixture was stirred in the dark at room temperature for 17 h. TLC showed full conversion of the donor (CyH / EtOAc 1 : 1, KMnO4). The solution was filtered through Celite® to remove solids and the filtrate was concentrated under reduced pressure. The residue was diluted with EtOAc (40 ml) and washed with saturated NaHCCh (6 x 20 ml), water and brine. The organic layer was dried over MgSCh, filtered and concentrated under reduced pressure to afford (2S,3R,4S,5S,6S)-2- (4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (109 mg, 0.225 mmol, 89 %) as a white solid.

[0172] *HNMR (400 MHz, CDC13) 8 9.96 (s, 1H), 8.29 (d, 7= 2.0 Hz, 1H), 8.07 (dd, 7= 8.6, 2.0 Hz, 1H), 7.50 (d, 7= 8.6 Hz, 1H), 5.45 - 5.37 (m, 2H), 5.35 - 5.24 (m, 2H), 4.33 (d, J= 8.4 Hz, 1H), 3.69 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H).

[0173] LC-MS (ESI): m / z = 501 [M+H2O]

[0174] Synthesis of (2S,3R,4S,5S,6S)-2-(4-(l-hydroxybut-3-yn-l-yl)-2-nitrophenoxy)-6 (methoxy carbonyl)-tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (SYB-BA2-060):

[0175] To a suspension of freshly activated zinc powder (HC1, H2O, MeOH, Et2O, high vacuum dry, 54 mg, 0.83 mmol, 8.0 eq.) in dry THF (2 mL, 0.4 m) was added allyl bromide (123 mg, 0.825 mmol, 8.0 eq.) at 0 °C under a nitrogen atmosphere. The reaction was warmed to roomtemperature and stirred for 1 h until all zinc was dissolved. The reaction solution was cooled to -15 °C and a solution of (2S,3R,4S,5S,6S)-2- (4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (50 mg, 0.103 mmol, 1.0 eq.) in THE (1 mL) was slowly added. The reaction mixture was stirred for 30 min at -15 °C and quenched by addition of a saturated aqueous NH4CI solution (20 mL). The aqueous solution was extracted with EtOAc (3*20 mL). The combined organic layers were washed with H2O (30 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (12g, 20 CV, CyH:EtOAc: 95:5 to 20:80) to afford (2S,3R,4S,5S,6S)-2-(4-(l-hydroxybut-3-yn-l-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)-tetrahydro-2H- pyran-3,4,5-triyl triacetate (14 mg, 1.16 mmol, 53 %).

[0176] ‘H NMR (400 MHz, CDCI3) 5 7.87 (ddd, J= 5.5, 2.2, 0.6 Hz, 1H), 7.58 (dddd, J= 8.6, 6.3, 2.2, 0.6 Hz, 1H), 7.37 (d, J= 8.6 Hz, 1H), 5.41 - 5.25 (m, 3H), 5.24 - 5.17 (m, 1H), 4.91 (t, J= 6.2 Hz, 1H), 4.24 - 4.18 (m, 1H), 3.74 (s, 3H), 2.70 - 2.57 (m, 2H), 2.13 (s, 3H), 2.11 (t, J= 2.6 Hz, 1H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (d, J= 3.8 Hz, 1H).

[0177] EC -MS (ESI): m / z = 541 [M+H2O]

[0178] Synthesis of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(l-(((4-nitrophenoxy)carbonyl)oxy)but-3-yn-l- yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (SYB-BA2-23):tri azaheptadec- 16-yn- 14-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3 ,4, 5-triyl triacetate (SYB-BA2-026):

[0184] To aa ssoolluuttiioonn ooff (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(l-(((4-nitrophenoxy)carbonyl)oxy)but-3-yn-l- yl)phenoxy)tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (60 mg, 0.87 mmol, 1 eq.), monomethylauristatin E (63 mg, 0.87 mmol, 1 eq.) and DIPEA (23 pL, 0.13 mmol, 1.5 eq.) and pyridine (0.43 mL, 0.87 mmol, 1 eq. 0.2 M in DMF) in DMF (4 mL) was added HOBT (14 mg, 0.43 mmol, 1.05 eq ).

[0185] The reaction mixture was stirred overnight. LCMS showed formation of product plus some SM MMAE remaining.

[0186] No purification was attempted. The crude reaction product was hydrolysed directly.

[0187] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)- l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7,10-diisopropyl-5,ll-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,l 1- tri azaheptadec- 16-yn- 14-yl)-2-nitrophenoxy)-3, 4, 5 -trihydroxytetrahydro-2H-pyran-2-carboxylic acid (SYB-BA1-34):

[0188] The reaction mixture (SYB-BA2-26) was diluted with MeOH (20 mL) and cooled to 0°C. To the resulting solution was added LiOH (8.8 eq., 1 M in H2O, 0.76 mL, 0.76 mmol) and the reaction was followed by TLC (DCM / MeOH 9 / 1). The resulting solution was quenched with HCOOH (20 eq., 1 M in H2O, 1.1 mL, 1.1 mmol) and concentrated. The reaction was diluted in water and extracted with DCM three times. LCMS of both organic and aqueous layers showed that the product stayed in the organic layer. The solvent was removed under reduced pressure. The residue in DMF was diluted with water and lyophilized overnight. The crude mixture was used for the click reaction. Purification at this stage is not necessary if the impurity is below 15%, since this impurity will be removed during the reverse phase purification of the final click reaction.General Procedure for the Click Reaction

[0189] To a solution of alkyne derivative (1.0 eq.) in DCM (0.05 M) was added the corresponding azide (1.3 eq.) followed by the addition of tetrakis(acetonitrile)copper(i)hexafluoro phosphate (1.5 eq.). The solution was stirred at room temperature and the reaction was monitored by LCMS. After 4 hours (SM was consumed as confirmed by LCMS), the reaction mixture was concentrated under reduced pressure to remove DCM. Then the crude product was diluted with DMF (0.05 M) and a solution of ethylenediaminetetraacetic acid di sodium salt dihydrate (EDTA.2Na.2H2O) (4 eq.) in water (0.15 M) was added at 0°C and stirring at room temperature was continued for 15 minutes. The reactioner- -) ,g - 1 -3 -u R -1,Bpe-2- - n2,1cmy 1 xh2- Y 2aTrR1,ilorS(.-)S y1(5 y3((-lxo d ox y o nre (ta(- -p- -2 y 2 0 p b dilh-iroitcw (-1H ,2- -)orraos1t--2aeni 3-7-orS((pc- 2et)iR1,rA)l)ld -oy 2si-in h2,9,6 moF yty(u ht ha -d-arw S ya1 -lrf% beort3-)1l0.-c1,p- s((y ( hted0 exesot-yxytu7- Hat-3-eurn -)2-ob)l2y- cu)RmidciNS()(l r-yd -choyestred d yo-r2,1di1,caC - h -)ohp R 1 5cA 4-)l1-irt-S(xart e (( -lilgnny- id5,(-o-etht -e2 y y p xis4il42- y -u -lo4,-))lxva )orobdeorr3-)Sy-org S((- p oi.zayp y 0srax1,1- dy).2iic-firdiruilt-)lo Snih q ie (-d-2-np o 3ypne7,dil rt-3.3- 0arss ,a eti2,orh S 1- p p oor4,orr5,1 )lyt1,y y4,,lu7-) p-wth H xtiR 3(p)l3 o -)m b-lcy Hcw uda1-o-3n-(- y yµ eshte2- oror s)ly-l2- 4 ) p x l(-oo 05 -)o dpadtn yhy- 6-rpne ,S(x o- yened u urop)te4 om-1-:))S ox h g p(-h42- ar76,o-orm2 -))l tcpdie2t-na0 R 3-lti1(S0y- yem h yc 0-5,n-1x ot mxeS ye1, -ordcateo d A a4,htnadece2-ahte t) diSnid)rin B- Slyma7,dilyh s oeCP3,m- -4teS 4,or irmed mm-1paS S22- rty1-caR y -g5eor-2 h)zai-6(x n o3(p5),( t b- ortld 2(-3- n4o,iohy-6 m(- y 3 -ni1 1tcte1-or4(- p -)nrm o 5( ma1,Aaem-ceb- 6ory p xulffo a-1)l8,5B-r1 d2-co(-y- -aB k) -)o o nc -)at lS x y6,o- eet52- Yiopths1 x Slcni enR 3 pade-)naoi r eha ep5,-lyorhzilS p 0o d-3o7htmazao S htipih1, r1,0r)lyirdiz4,Sten-esrp Spleo y2vy 7,n-0- of-t-a-3,m-2-eo Aer2- 1 1,5 S2-)lyelr eSere4,h xoB- una(p8, -2(y -4 Rp-ird t Cecor5- Nx d o1-htw3(1- -2 PSoorpl ax nfhten yt)S(- y 4 x1, -5pyoia).o - m 6deM(t-ord9,63-lanehd- q -11-cC 6- y -l2rep- 3 1,e. n -)ceejL)niy Sh- yh Ane1-2-0oito n d ayb 6,1-)teB- g e y o1,lul i tpltdceRe5,RmiB Y h x o x r o oosma)eh S2,d- Stg dirtml ar-imrµ yi-zdf4,S1 1( niysn S((1,h2 9a2air5diaocw - c3,(- o 11,3, -n 3 -l all-)9, at-1 6 g pw(esSrn2(-)ypcilo R1,F -lmoTor8,utoi]Rory2,t0 x]S yh 44 92,p o o 19 1( te( .) ]2pl5 y 9 -naxicmar1 R f0[1s((iibr1(dac0[(- 3midi% 1ex dca850[h poid

[0193] BA2-28 assumed 87 umol, 1.0 eq.) and N-(5-azidopentyl)-2-bromoacetamide (28.2 mg, 113 pmol, 1.3 eq.) in anhydrous DCM (6 mL) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (48.7 mg, 131 pmol, 1.5 eq.). The reaction mixture was stirred overnight. DCM was concentrated under reduced pressure, the residue was dissolved in DMF (1.5 mL) and treated with a solution of sodium 2, 2'-(ethane-l,2-diylbis((carboxymethyl)azanediyl))di acetate dihydrate (128 mg, 0.350 mmol, 4 eq.) in H2O (1.5 mL) at 0°C. The reaction was stirred for 15 min and the reaction mixture was directly injected to the reverse phase column (40 gm, 15pm). The crude was purified using ACN (5 / 95-95 / 5, 20CV) in 0.1% FA with water. The pure fractions (confirmed by LCMS) were lyophilized to afford the desired compound as a white solid (42 mg, 31 pmmol, 35% over 3 steps).

[0194] JHNMR (400 MHz, DMSO) 5 8.24 (m, 1H), 7.94 - 7.74 (m, 2H), 7.66 - 7.52 (m, 1H), 7.32 - 7.25 (m, 5H), 7.18 (m, 1H), 5.96 - 5.75 (m, 1H), 5.56 - 5.15 (m, 5H), 4.84 - 4.08 (m, 6H), 4.08 - 3.91 (m, 3H), 3.82 (m, 2H), 3.59 (s, 1H), 3.27 - 3.11 (m, 8H), 3.02 (m, 4H), 2.96 - 2.65 (m, 3H), 2.28 (s, 1H), 2.20 - 1.92 (m, 3H), 1.80-1.72 (m, 5H), 1.61 - 1.12 (m, 10H), 1.07 - 0.96 (m, 8H), 0.92 - 0.73 (m, 18H), 0.72 - 0.57 (m, 4H), 0.46 (d, J= 6.5 Hz, 1H).

[0195] LCMS Purity was 99 %.

[0196] Note: the sequence of MMAE coupling, Ac deprotection and click reaction can be done without reverse phase purification after MMAE coupling or acetyl deprotection. 40 mg of final product with purity > 95 % (44 % over 3 steps) was obtained from 60 mg activated benzylic alcohol.

[0197] LC-MS (ESI): m / z = 1377 [M+H]+

[0198] 2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S, 10S)-15-(l-(5-(2-bromoacetamido)pentyl)-lH-l,2,3- triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2- yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7,10-diisopropyl-5,ll- dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,l l-triazapentadecan-14-yl)-2-nitrophenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid (SYB-BA2-63- SPC-BA-007):

[0199] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(l-(5-(2-bromoacetamido)pentyl)-lH-l,2,3- triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2- yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7,10-diisopropyl-5,ll- dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,l l-triazapentadecan-14-yl)-2-nitrophenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid from reactions: (SYB-BA2-06, 31, 35, 37, 44, 53, 55) were combined in ACN / H2O (1 / 1 : 15 / 15 mL) and lyophilized for two days to afford (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(l-(5-(2-bromoacetamido)pentyl)-lH-l,2,3- triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-

[0200] yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7, 10-diisopropyl-5,ll-dimethyl-6,9,12-trioxo-2, 13-dioxa-5, 8, 11 -triazapentadecan- 14-yl)-2-nitrophenoxy)-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid 3,4,5-trihydroxytetrahydro-2H-pyran- 2-carboxylic acid (507 mg, 368 pmol).Linker for SPC-BA-007

[0201] General procedure A for the azide substitution: To a solution of Br compound (1 eq.) in DMF (0.2 m) was added sodium azide (1.5 eq) and the reaction mixture was stirred at 70 °C for 16 h. It was then washed with saturated aqueous NaHCCh solution and EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude products were used without further purification.

[0202] General procedure B for Phthalimide deprotection: To a solution of phthalimide compound ( 1 eq.) in THF (0.15 M) was added hydrazine monohydrate (40 eq.). The reaction mixture was stirred at room temperature for 4 hrs. THF was removed under reduced pressure and the residue was washed with saturated aqueous NaHCO? and EtOAc. The combined organic layers were dried, filtered, and concentrated. The crudeproducts were used without further purification.

[0203] Modified procedure B for phthalimide deprotection: To a solution of phthalimide compound (1 eq.) in THE (0.2 M) was added hydrazine monohydrate (3 eq.). The reaction mixture was stirred at room temperature overnight. The phthalic hydrazide was removed by filtration and washed with THE. The solvent was removed under reduced pressure to afford the desired amine. The crude products were used without further purification.

[0204] General procedure D for bromoacetylbromide substitution: To a solution of amine (1 eq.) in DCM (0.20 M) was added K2CO3 (1.5 eq.) in water (0.5 mL). Then 2-bromoacetylbromide (1.1 eq.) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 15 hrs. It was then washed with water and EtOAc. The combined organic layers were dried, filtered, and concentrated and purified by column chromatography.

[0205] 2-(5-azidopentyl)isoindoline-l, 3-dione (SYB-BA2-19). Following the general procedure A for azide substitution, A solution of 2-(5-bromopentyl)isoindoline-l, 3-dione (3.00 g, 10.1 mmol, 1 eq.) and sodium azide (988 mg, 15.2 mmol, 1.5 eq.) in dimethyl formamide (20 mL, 0.5M) was stirred at 70 °C overnight. The reaction was cooled to room temperature, diluted with brine (100 mL) and extracted three times with ethyl acetate (3 x 50 mL). The combined organic layers were washed three times with water, dried overNa2SO4, filtered and concentrated, to afford 2-(5-azidopentyl)isoindoline- 1, 3-dione (2.58 g, 10.0 mmol, 98% yield).

[0206] 1H NMR (400 MHz, CDCI3) 5 7.91 - 7.78 (m, 2H), 7.76 - 7.65 (m, 2H), 3.78 - 3.62 (m, 2H), 3.27 (t, J = 6.9 Hz, 2H), 1.78 - 1.60 (m, 4H), 1.50 - 1.37 (m, 2H).

[0207] A-(5-azidopentyl)-2-bromoacetamide (SYB-BA2-18), Following the general procedure D for bromoacetylbromide substitution, To a solution of 5 -azidopentan- 1 -amine (500 mg, 3.9 mmol, 1 eq.) in DCM (20 mL) was added potassium carbonate (1.10 g, 7.8 mmol, 2 eq.), and 2- bromoacetylbromide (0.38 mL, 4.3 mmoL, 1.1 eq.) dropwise at 0 °C. The reaction was stirred overnight, then washed with water and EtOAc. The combined organic layers were dried over sodium sulfate, filtered and concentrated.

[0208] Purification of the residue by MPLC (SiO2) (20g, 20 CV) (CyH:EtOAc: 100:0 to 40:60) afforded N-(5-azidopentyl)-2-bromoacetamide (639 mg, 2.57 mmol, 66%).

[0209] Analytical Data. TLC [SiO2] (CyH:EtOAc:50:50). Rf= 0.40 (UV, KMnO4)

[0210] JHNMR (400 MHz, CDCI3) 5 6.51 (s, 1H), 3.88 (s, 2H), 3.34 - 3.23 (m, 4H), 1.68 - 1.54 (m, 4H), 1.47 - 1.37 (m, 2H).Synthesis of SPC-BA-017

[0211] (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate SYB-BA2-066:

[0212] To a solution of (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5- triyl triacetate (100 mg, 0.252 mmol, 1.0 eq.) in dry MeCN (2.5 mL, 0.1 m) was added 4-hydroxy-3- nitrobenzaldehyde (63 mg, 0.378 mmol, 1.5 eq.) and silver oxide (87 mg, 0.38 mmol, 1.5 eq.) at room temperature under a nitrogen atmosphere. The resulting reaction mixture was stirred in the dark at room temperature for 17 h. TLC showed full conversion of the donor (cHex / EtOAc 1 : 1, KMnCU). The solution was filtered through Celite® to remove solids and the filtrate was concentrated under reduced pressure. The residue was diluted with EtOAc (40 ml) and washed with saturated NaHCCh (6 x 20 ml), water and brine. The organic layer was dried over MgSCh, filtered and concentrated under reduced pressure ttoo afford (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (109 mg, 0.225 mmol, 89 %) as a white solid.

[0213] Tl NMR (400 MHz, CDCh) 8 9.96 (s, 1H), 8.29 (d, J- 2.0 Hz, 1H), 8.07 (dd, J- 8.6, 2.0 Hz, 1H), 7.50 (d, J= 8.6 Hz, 1H), 5.45 - 5.37 (m, 2H), 5.35 - 5.24 (m, 2H), 4.33 (d, J= 8.4 Hz, 1H), 3.69 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H).

[0214] 2S,3R,4S,5S,6S)-2-(4-(l-hydroxybut-3-yn-l-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)- tetrahydro-2H-pyran-3,4,5-triyl triacetate SYB-BA1-060:

[0215] To a suspension of freshly activated zinc powder (HC1, H2O, MeOH, Et2<), high vacuum dry, 54 mg, 0.83 mmol, 8.0 eq.) in dry THF (2 mL, 0.4 m) was added allyl bromide (123 mg, 0.825 mmol, 8.0 eq.) at 0 °C under a nitrogen atmosphere. The reaction was warmed to room temperature and stirred for Ih until all zinc was dissolved. The reaction solution was cooled to -15 °C and a solution of(2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (50 mg, 0.103 mmol, 1.0 eq.) in THF (1 mL) was slowly added. The reaction mixture was stirred for 30 min at -15 °C and quenched by addition of a saturated aqueous NH4CI solution (20 mL). The aqueous solution was extracted with EtOAc (3*20 mL). The combined organic layers were washed with H2O (30 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (12g, 20 CV, CyH:EtOAc: 95:0 to 20:80) ttoo afford (2S,3R,4S,5S,6S)-2-(4-(l-hydroxybut-3-yn-l-yl)-2-nitrophenoxy)-6- (methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyl triacetate (14 mg, 1.16 mmol, 53 %).

[0216] JH NMR (400 MHz, CDCI3) 5 7.87 (ddd, J= 5.5, 2.2, 0.6 Hz, 1H), 7.58 (dddd, J= 8.6, 6.3, 2.2, 0.6 Hz, 1H), 7.37 (d, J= 8.6 Hz, 1H), 5.41 - 5.25 (m, 3H), 5.24 - 5.17 (m, 1H), 4.91 (t, J= 6.2 Hz, 1H), 4.24 - 4.18 (m, 1H), 3.74 (s, 3H), 2.70 - 2.57 (m, 2H), 2.13 (s, 3H), 2.11 (t, J = 2.6 Hz, 1H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (d, J= 3.8 Hz, 1H).

[0217] (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(l-(((4-nitrophenoxy)carbonyl)oxy)but-3-yn-l-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate SYB-BA1-94 or SYB-BA2-23:

[0218] To a solution of (2S,3R,4S,5S,6S)-2-(4-(l-hydroxybut-3-yn-l-yl)-2-nitrophenoxy)-6- (methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyl triacetate, SYB-BA1-060, (375 mg, 0.71 mmol, 1.0 eq.), 4-Nitrophenyl chloroformate (361 mg, 1.79 mmol, 2.5 eq.) in dichloromethane (8 mL, 0. 1 m) at 0 °C was added pyridine (580 pL, 7.16 mmol, 10 eq.), and the reaction was stirred for 4 hours at room temperature. The reaction was diluted with EtOAc (100 mL) and washed with aqueous saturated NaHCOs. The aqueous layer was extracted three times with EtOAc The combined organic layers were dried, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CyH / EtOAc 5 to 60 %) to afford (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro- 4-( 1 -(((4-nitrophenoxy)carbonyl)oxy)but-3 -yn- 1 -yl)phenoxy)tetrahydro-2H-pyran-3 ,4,5 -triyl triacetate (410 mg, 0.590 mmol, 83%).

[0219] Rf- 0.30 product (KMnO4)

[0220] JHNMR (400 MHz, CDCh) 5 8.35 - 8.27 (m, 2H), 7.96 (d, J= 2.2 Hz, 1H), 7.67 (dd, J= 8.5, 2.3 Hz, 1H), 7.47 - 131 (m, 3H), 5.83 (t, J = 6.5 Hz, 1H), 5.47 - 5.22 (m, 4H), 4.26 (d, J = 8.8 Hz, 1H), 3.76 (s, 3H), 3.06 - 2.85 (m, 2H), 2.15 (s, 3H), 2.12 (t, J= 2.6 Hz, 1H), 2.10 (s, 3H), 2.06 (s, 3H).

[0221] (2S,3R,4S,5S,6S)-2-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((l S,2R)- 1 -hydroxy- 1 -phenylpropan-2-yl)amino)- 1 -methoxy -2 -methyl-3 -oxopropyl)pyrrolidin- 1 -yl)-2- oxoethyl)-7,10-diisopropyl-5,l l-dimethyl-6,9,12-trioxo-2, 13-dioxa-5,8, l l-triazaheptadec-16-yn-14- yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, SYB-BA1-029 or SYB-BA2-026:

[0222] To aa ssoolluuttiioonn of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(l-(((4-nitrophenoxy)carbonyl)oxy)but-3-yn-l- yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (110 mg, 0.16 mmol, 1 eq.), monomethylauristatin E (115 mg, 0.160 mmol, 1.0 eq.) and DIPEA (61 pL, 0.35 mmol, 2.2 eq.) and pyridine (0.13 pL, 0.16 mmol, 1.0 eq.) in DMF (3.2 mb, 0.05 m) were added HOBT (26 mg, 0.17 mmol, 1.05 eq.).

[0223] The reaction mixture was directly injected to the reverse phase column and purified by reverse phase column chromatography using 5- 70% methanol in 0.1% FA with water to afford (2S,3R,4S,5S,6S)-2-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((l S,2R)-1- hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7,10-diisopropyl-5,ll-dimethyl-6,9, 12- tri oxo-2, 13 -dioxa-5, 8, 11 -triazaheptadec- 16-yn- 14-yl)-2-nitrophenoxy)-6-(methoxy carbonyl )tetrahydro-2H-pyran-3, 4, 5 -triyl triacetate (216 mg, 0.170 mmol, 60 %) as a white solid.

[0224] *HNMR (400 MHz, MeOD) 5 7.98 - 7.57 (m, 2H), 7.56 - 7.13 (m, 6H), 5.89 - 5.73 (m, 1H), 5.58 - 5.36 (m, 2H), 5.23 (q, J= 8.6 Hz, 2H), 4.71 (d, J = 8.7 Hz, 1H), 4.65 - 4.46 (m, 3H), 4.33 - 4.01 (m, 3H), 3.80 - 3.63 (m, 4H), 3.46 - 3.33 (m, 9H), 3.29 -3.23 (m, 3H), 3.16 -2.75 (m, 6H), 2.55 - 2.30 (m, 3H), 2.30 - 1.99 (m, 11H), 1.98 1.75 (m, 3H), 1.59 (s, 1H), 1.41 (s, 1H), 1.23 1.08 (m, 6H), 0.99 - 0.83 (m,17H), 0.69 - 0.48 (m, 2H)

[0225] LC-MS (ESI): m / z = 1268 [M+H]+

[0226] 2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l- methoxy-2-m ethyl -3-oxopropyl )pyrrolidin- 1 -yl)-2-oxoethyl)-7, 10-diisopropyl-5, 11 -dimethyl-6, 9,12-trioxo-2,l 3-dioxa-5,8, 11 -triazaheptadec- 16-yn-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, SYB-BA1-34:

[0227] To a solution of (2S,3R,4S,5S,6S)-2-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l- phenylpropan-2-yl)amino)- 1 -methoxy-2-methyl-3 -oxopropyl)pyrrolidin- 1 -yl)-2-oxoethyl)-7, 10-diisopropyl-5, 11 -dimethyl-6, 9, 12-trioxo-2, 13- dioxa-5,8,ll-triazaheptadec-16-yn-14-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, SYB-BA1-029 or SYB-BA2-026, (200 mg, 158 pmmol, 1.0 eq.) in MeOH (6 mL, 0.03 m) cooled at 0°C, was added dropwise a cold solution (0°C) of l.iOIMEO (58 mg, 1.4 mmol, 8.8 eq.) in H2O (6 mL). Stirring was continued for 15 minutes at 0°C (followed by TLC DCM:MeOH 9:1) and the solution was neutralized with Amberlite™ Weakly acidic Cation exchanger, hydrogen form, during 15 minutes (pH followed on paper) and filtered. The aqueous layer was freeze dried overnight to afford (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((lS,2R)- l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7,10-diisopropyl-5,ll-dimethyl- 6,9,12-trioxo-2,13-dioxa-5,8,ll-triazaheptadec-16-yn-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid whichcontained 8% of impurity by HPLC (Method ACN-high mass-2000: RT: 3.79 min, m / z= 1109 [M+H] ). The impure product was purified by reverse phase column chromatography (40 g, 15pm) using MeOH in 0.1% FA with water to afford (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)- sec-butyl)-3 -(2-((S)-2-(( 1 R,2R)-3 -((( 1 S,2R)- 1 -hydroxy- 1 -phenyl propan-2 -yl)amino)- 1 -methoxy-2- methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7,10-diisopropyl-5,ll-dimethyl-6,9, 12-trioxo- 2,13-dioxa-5,8,l l-triazaheptadec-16-yn-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid (89 mg, 79 pmmol, 50%, containing less than 3% impuritu) and (56 mg, 50 pmmol, 31%, containing less than 10% impurity).

[0228] Purification at this stage is not necessary ifthe impurity level is below 15%, since the impurity will be removed during the reverse phase purification of the final click reaction.

[0229] JHNMR (400 MHz, MeOD) 8 7.97 - 7.85 (m, 1H), 7.69 (m 1H), 7.50 - 7.17 (m, 6H), 5.80 (d, J= 9.6 Hz, 1H), 5.28 - 5.07 (m, 1H), 4.75 - 4.42 (m, 2H), 4.39 - 3.43 (m, 10H), 3.35 (m, 6H), 3.30 - 3.22 (m, 5H), 3.14 - 2.69 (m, 6H), 2.62 - 2.07 (m, 5H), 2.05 - 1.51 (m, 5H), 1.41 (s, 2H), 1.31 - 0.26 (m, 26H).

[0230] LC-MS (ESI): m / z = 1128 [M+H]+LC-MS (ESI): m / z = 1128 [M+H]+General Procedure for the Click Reaction

[0231] To a solution of alkyne derivative (1.0 eq.) in DCM (0.05 M) was added the corresponding azide (1.3 equiv.) followed by the addition of tetrakis(acetonitrile)copper(i)hexafluoro phosphate (1.5 eq.). The solution was stirred at room temperature and the reaction was monitored by LCMS until the desired mass was found. After 4 hours (SM was consumed as confirmed by LC-MS), the reaction mixture was concentrated under reduced pressure to remove the DCM. Then the crude product was diluted with DMF and a solution of ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA.2Na.2H2O) (4 eq.) in water was added at 0°C and stirring at room temperature was continued for 15 minutes. Then the reaction mixture was directly injected to the reverse phase column (25 gm) and the crude product was purified using ACN in 0.1% FA in water. The pure fraction (confirmed by LCMS) was lyophilized to afford the desired compound as a white solid.

[0232] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(l-(l-bromo-2-oxo-6,9,12,15,18,21,24,27- octaoxa-3-azanonacosan-29-yl)-lH-l,2,3-triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3- ((( 1 S,2R)- 1 -hydroxy- 1 -phenylpropan-2-yl)amino)- 1 -methoxy -2-methyl-3 -oxopropyl)pyrrolidin- 1 - yl)-2-oxoethyl)-7,10-diisopropyl-5,ll-dimethyl-6, 9, 12-trioxo-2,13-dioxa-5, 8, 11-tri azapentadecan- 14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, SYB-BA1-111 or SPC-BA-017

[0233] By following the general procedure for the click reaction (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2- ((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7,10- diisopropyl-5, 1 l-dimethyl-6,9, 12-trioxo-2, 13-dioxa-5,8, 1 l-triazaheptadec-16-yn-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid (40 mg, 35 pmol, 1 eq.) and JV-(26-azido-3, 6, 9, 12, 15, 18,21, 24-octaoxahexacosyl)-2 -bromoacetamide (25 mg, 46 pmol, 1.3 eq.) gave the desired product as a white solid (9.7 mg, 5.8 pmmol, 16%).

[0234] 'H NMR (400 MHz, DMSO) 5 8.36 - 8.32 (m, 1H), 7.92 - 7.84 (m, 1H), 7.75 - 7.69 (m, 1H), 7.40 - 7.20 (m, 6H), 7.21 - 7.15 (m, 1H), 5.43 - 5.33 (m, 3H), 5.26 - 5. 18 (m, 2H), 4.55 - 4.28 (m, 6H), 4. 10 - 3.89 (m, 2H), 3.86 (s, 2H), 3.76 (s, 3H), 3.71 - 3.31 (m, 36H), 3.27 - 3.21 (m, 10H), 3.06 - 2.79 (m, 1H), 2.78 - 2.72 (m, 1H), 2.44 - 1.90 (m, 2H), 1.72 - 1.62 (m, 1H), 1.35 - 1.15 (m, 3H), 1.10 - 0.94 (m, 10H), 0.93 - 0.55 (m, 24H), 0.47 (d, J= 6.6 Hz, 1H).

[0235] LCMS Purity was 96 %.

[0236] LC-MS (ESI): m z = 1687 [M+H]+Linker for SPC-BA-017

[0237] Ar-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)-2-bromoacetamide SSA-BA1-164:

[0238] To a solution of 26-azido-3,6,9,12,15,18,21,24-octaoxahexacosan-l-amine (100 mg, 0.228 mmol, 1.0 eq.) in dry DCM (6 mL) was added K2CO3 (47 mg, 0.34 mmol, 1.5 eq.) followed by the addition of 2 -bromoacetyl bromide (83 mg, 0.41 mmol, 1.8 eq.). The reaction mixture was stirred for 3 h at room temperature. After completion of the reaction, it was diluted with water and extracted twice with EtOAc. The combined organic layers were washed with water, filtered, and concentrated to afford the desired compound as a pale-yellow liquid (110 mg, 86%). The crude product was used without further purification.

[0239] ‘H NMR (400 MHz, CDCI3) 5 3.93 (s, 2H), 3.89 (s, 13H), 3.71 - 3.62 (m, 24H).

[0240] Synthesis of SPC-BA-019

[0241] Synthesis steps for SPC-BA-019 up to the general procedure of the click reaction are carried out as set forth above for SPC-BA-007 and SPC-BA-017.

[0242] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S, 10S)-15-(l-(2-(2-bromoacetamido)ethyl)-lH-l,2,3-triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)arnino)-l-methoxy-2-rnethyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-7,10- diisopropyl-5, 11 -dimethyl-6, 9, 12-tri oxo-2, 13-di oxa-5, 8, 11 -tri azapentadecan- 14-yl)-2-nitrophenoxy)-3, 4, 5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid, SYB-BA1-132 SPC-BA-019:Linker for SPC-BA-019

[0247] 2-(2-azidoethyl)isoindoline-l, 3-dione SSA-BA1-127

[0248] To a stirred solution of 2-(2-bromoethyl)isoindoline-l, 3-dione (2.5 g, 9.8 mmol, 1.0 eq.) in DMF (49 mL, 0.2 m) was added sodium azide (0.96 g, 1.5 eq., 15 mmol) and the resultant reaction mixture was stirred at 70 °C overnight. After completion of the reaction, aqueous 10% LiCl solution was added, and the mixture was extracted twice with EtOAc. The combined organic layers were washed with water, filtered, and concentrated to afford the desired compound as an off-white solid (2.01 g, 94%). The crude product was used without further purification.

[0249] XH NMR (500 MHz, CDCh) 5 7.90 - 7.83 (m, 2H), 7.76 - 7.71 (m, 2H), 3.90 (t, J= 6.1 Hz, 2H), 3.59 (t, J= 6.1 Hz, 2H).

[0250] 2-azidoethan-l-amine SSA-BA1-131

[0251] To a solution of 2-(2-azidoethyl)isoindoline-l, 3-dione (2.01 g, 9.30 mmol, 1 eq.) in THF (46.5 mL, 0.2 molar) was added hydrazine hydrate (18.6 g, 18.1 mL, 372 mmol, 40 eq.) and the resultant reaction mixture was stirred at room temperature for 4 hours. After completion of the reaction, THF was removed (staying above 250 mbar) and the residue was washed with 10% LiCl and extracted with diethyl ether. The combined organic layers were dried, filtered, and concentrated (staying above 250mbar) to afford the desired compound as a colorless oil (800 mg, crude, containing THF and diethyl ether as a solvent impurity).

[0252] Due to the volatile nature of the compound, it was difficult to remove the residual solvent and it was used in the next step.

[0253] Ar-(2-azidoethyl)-2-bromoacetamide SSA-BA1-134

[0254] A solution of 2-bromoacetylbromide (0.15 mL, 1.5 mmol, 2.2 eq.) in dry THF (3 mL) was added dropwise to the mixture of 2-azidoethan-l -amine (65 mg, 0.75 mmol, 1.0 eq.) and TEA (0.24 mL,1.7 mmol, 2.5 eq.) in dry THF (4.5 mL, 0.2 m) at 0 °C. The reaction mixture was stirred for 1.3 h at room temperature. The excess of acid bromide was quenched by the addition of methanol (1 mL). The formed triethylammonium bromide salt was filtered off and the solvent was removed under reduced pressure. The crude product was dissolved in dichloromethane and washed three times with a saturated ammonium chloride solution and two times with distilled water. The combined organic layers were dried, filtered, and concentrated to afford a pale brown oil crude product. The residue was purified by MPLC (SiCL, CyH / EtOAc 0 to 45 %) to afford the desired compound as a pale-yellow oil (65 mg, 42%).

[0255] JHNMR (400 MHz, CDC13) 6 6.76 (s, 1H), 3.90 (s, 2H), 3.48 (d, J= 4.1 Hz, 4H).LARGE SCALE CONJUGATION TO ANTIBODIES

[0256] Dilute TCEP to 5 mM: 5 ul of 0.5 M TCEP into 495 ul of SPC EDTA buffer (50 mM Tris pH8.5, 150 mM NaCl, 20 mM EDTA.

[0257] Perform conjugation in a 1.7 ml tube1. Aliquot 97 ul of SPC EDTA buffer2. Aliquot 847 ul of antibody at 5.9 mg / ml3. Slowly mix in 22 ul TCEP4. Place tube in shaker at 25 °C, 300 rpm for 1 hour5. Place on ice for 2 min6. Slowly mix in 33.3 ul linker payload (SPC004, SPC007, or SPC008)7. Place tube in shaker at 25 °C, 300 rpm for 2 hours8. Place on iceExemplary Anti-Cancer Agents

[0258] In some embodiments, an immunoconjugate comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine and are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0259] Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relatively accessible to prepare by fermentation or chemical modification or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation through non-disulfide linkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[0260] Certain maytansinoids suitable for use as maytansinoid drug moieties are known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see, e.g., Yu et al., PNAS, vol. 99, pp. 7968-7973, 2002). Maytansinoids may also be prepared synthetically according to known methods.

[0261] Exemplary maytansinoid drug moieties include, but are not limited to, those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared, for example, by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl)+ / -C- 19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared, for example, by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-dem ethoxy, C-20- acyloxy ( — OCOR), / dechloro (U.S. Pat. No. 4,294,757) (prepared, for example, by acylation using acyl chlorides), and those having modifications at other positions of the aromatic ring.

[0262] Exemplary maytansinoid drug moieties also include those having modifications such as: C-9- SH(U.S. Pat. No. 4,424,219) (prepared, for example, by the reaction of maytansinol with EES or P2S5); C-14-alkoxymethyl(demethoxy / CH2OR)(U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CI EOAc) (U.S. Pat. No. 4,450,254) (prepared, for example, from Nocardia), C- 15 -hydroxy / acyloxy (U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion of maytansinol by Streptomyces) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929)(for example, isolated from Trewia nudlfloray, C-18-N-dem ethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, for example, by the demethylation of maytansinol by Streptomyces),' and 4,5- deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by the titanium trichloride / LAH reduction of maytansinol).

[0263] Many positions on maytansinoid compounds are useful as the linkage position. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. In some embodiments, the reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In some embodiments, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.

[0264] Maytansinoid drug moieties include those having the structure:where the wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to a linker of an antibody-drug conjugate (ADC). Each R may independently be H or a Ci-Ce alkyl. The alkylene chain attaching the amide group to the sulfur atom may be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020; Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; and Liu et al., Proc. Nall. Acad. Sei. USA, vol. 93, pp. 8618-8623, 1996).

[0265] All stereoisomers of the maytansinoid drug moiety are contemplated for the ADC of the disclosure, i.e. any combination of R and S configurations at the chiral carbons (U.S. Pat. No. 7,276,497; U.S. Pat. No. 6,913,748; U.S. Pat. No. 6,441, 163; U.S. Pat. No. 633,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al., (2006), J. Med. Chem. 49:4392-4408. In some embodiments, the maytansinoid drug moiety has the following stereochemistry:

[0266] Exemplary embodiments of maytansinoid drug moieties include, but are not limited to, DM1;DM3; and DM4 having the structures.wherein the wavy line indicates the covalent attachment of the sulfur atom of the drug to a linker (L) of an ADC.

[0267] Exemplary antibody-drug conjugates where DM1 is linked through a BMPEO linker to a thiol group of the antibody have the structure and abbreviation:where Ab is an antibody; n is 0, 1, or 2; and p is 1 to about 20 In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

[0268] Immunoconjugates containing maytansinoids, methods of making the same, and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020 and 5,416,064; US 2005 / 0276812 Al; and European Patent EP 0 425 235 Bl. See also Liu et al., Proc. Natl. Acad. Sci. USA, vol. 93, pp. 8618-8623, 1996; and Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992.

[0269] In some embodiments, antibody-maytansinoid conjugates may be prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020. In some embodiments, anADC with an average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody. In some instances, even one molecule of toxin / antibody is expected to enhance cytotoxicity relative to the use of naked antibody.

[0270] Exemplary linking groups for making antibody-maytansinoid conjugates include, for example, those described herein and those disclosed in U.S. Pat. No. 5,208,020; EP Patent 0 425 235 Bl; Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992; US 2005 / 0276812 Al; and US 2005 / 016993 Al.

[0271] Anti-cancer agents include dolastatins, auristatins, and analogs and derivatives thereof (U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; U.S. Pat. No. 5,767,237; and U.S. Pat. No 6,124,431). Auristatins are derivatives of the marine mollusk compound dolastatin-10. While not intending to be bound by any particular theory, dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al, Antimicrob. Agents and Chemother., vol. 45, pp. 3580-3584, 2001) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob. Agents Chemother., vol. 42, pp. 2961-2965, 1998). The dolastatin / auri statin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02 / 088172; Doronina et al., Nature Biotechnology, vol. 21, pp. 778-784, 2003; Francisco et al., Blood, vol. 102, pp. 1458-1465, 2003).

[0272] Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties DE and DE, disclosed in U.S. Pat. No. 7,498,298 and U.S. Pat. No. 7,659,241wherein the wavy line of DE and DF indicates the covalent attachment site to an antibody or antibody-linker component, and independently at each location:R2is selected from H and Ci-Cg alkyl;R3is selected from H, Ci-Cs alkyl, Cg-Cg carbocycle, aryl, Ci-Cg alkyl-aryl, Ci-Cg alkyl-(Cg-Cg carbocycle), Cg-Cgheterocycle and Ci- Cg alkyl-(C3-Cg heterocycle);R4is selected from H, Ci-Cg alkyl, Cg-Cg carbocycle, aryl, Ci-Cg alkyl-aryl, Ci-Cg alkylRCg-Cg carbocycle), Cg-Cgheterocycle and Ci-Cg alkyl-(Cg-Cg heterocycle);R5is selected from H and methyl; or R4and R5jointly form a carbocyclic ring and have the formula (CRaRb)n— wherein Raand Rbare independently selected from H,Ci-Cg alkyl and Cg-Cg carbocycle and n is selected from 2, 3, 4, 5 and 6;R6is selected from H and Ci-Cg alkyl;R7is selected from H, Ci-Cs alkyl, Cg-Cg carbocycle, aryl, Ci-Cs alkyl-aryl, Ci-Cs alkyl-(C3- Cg carbocycle), C3-Cxheterocycle and Ci-Cs alkyl-(C3-Cs heterocycle); each R8is independently selected from H, OH, Ci-Cg alkyl, C3-Cs carbocycle and 0 — (Ci- Cg alkyl);R9is selected from H and Ci-Cs alkyl,R1(lis selected from aryl or C3-Cs heterocycle;Z is 0, S, NH, or NR12, wherein R12is Ci-Cs alkyl;R11is selected from H, C1-C20 alkyl, aryl, C3-G heterocycle, — (R130)m— R14, or — (R13O)m-CH(R15)2; m is an integer ranging from 1-1000;R13is C2-C8alkyl;R14is H or Ci-Cg alkyl; each occurrence of e is independently H, COOH, (CH2)n- N(R16)2, — (CH2)n— SO3H, or— (CH2)n— SO3— Ci-Cg alkyl; each occurrence of e is independently H, Ci-Cg alkyl, or — (CH2)n— COOH;R18is selected from — C(R8)2— C(R8)2-aryl, — C(R8)2— C(R8)2— (G-G heterocycle), and C(R8)2— C(R8)2— (C3-C8carbocycle); and n is an integer ranging from 0 to 6.

[0273] In one embodiment, R3, R4and R7are independently isopropyl or sec-butyl and R5is -H or methyl. In an exemplary embodiment, R3and R4are each isopropyl, R5is — H, and R7is sec-butyl.

[0274] In yet another embodiment, R2and R6are each methyl, and R9is - H.

[0275] In still another embodiment, each occurrence of R8is — OCH3.

[0276] In an exemplary embodiment, R3and R4are each isopropyl, R2and R6are each methyl, R5is — H, R7is sec-butyl, each occurrence of R8is — OCH3, and R9is — H.

[0277] In one embodiment, Z is — O — or — NH — .

[0278] In one embodiment, R10is aryl.

[0279] In an exemplary embodiment, R10is -phenyl.

[0280] In an exemplary embodiment, when Z is — O -, R11is — H, methyl or t-butyl.

[0281] In one embodiment, when Z is — NH, R11is CH(R15)2, wherein R15is — (CH2)n— N(R16)2, and R16is — Ci-Cg alkyl or — (CH2)n— COOH.

[0282] In another embodiment, when Z is — NH, R11 is — CH(R15)2, wherein R15is — (CH2)n— SO3H.

[0283] An exemplary auristatin embodiment of formula DE is MMAE, wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody-drug conjugate:

[0284] An exemplary auristatin embodiment of formula DE is MMAF, wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody-drug conjugate:

[0285] Other exemplary embodiments include monomethylvaline compounds having phenylalanine carboxy modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008848) and monomethylvaline compounds having phenylalanine sidechain modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008603).

[0286] Nonlimiting exemplary embodiments of ADCs comprising MMAF and various linker components also include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Immunoconjugates comprising MMAF attached to an antibody by a linker that is not proteolytically cleavable have been shown to possess activity comparable to immunoconjugates comprising MMAF attached to an antibody by a proteolytically cleavable linker(Doronina et al, Bioconjugate Chem., vol. 17, pp. 114-124, 2006). In certain such embodiments, drug release is believed to be effected by antibody degradation in the cell.

[0287] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more ammo acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method (see, e.g., E. Schroder and K. Liibke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press). Auristatin / dolastatin drug moieties may, in some embodiments, be prepared according to the methods of: U.S. Pat. No. 7,498,298; U.S. Pat No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al, J. Am. Chem. Soc., vol. Ill, pp. 5463-5465, 1998; Pettit et al., Anti -Cancer Drug Design, vol. 13, pp. 243-277, 1998; Pettit et al., Synthesis, vol. 6, pp. 719-725, 1996; Pettit et al., J. Chem. Soc. Perkin Trans, vol. 15, pp. 859-863, 1996; and Doronina , Nat. Biotechnol., vol. 21, pp. 778-784, 2003.

[0288] In some embodiments, auristatin / dolastatin drug moieties of formulas DE such as MMAE, and DF, such as MMAF, and drug-linker intermediates and derivatives thereof, such as MC- MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methods described in U.S. Pat. No. 7,498,298; Doronina et al., Bioconjugate Chem., vol. 17, pp. 114-124, 2006; and Doronina et al., Nat. Biotech., vol. 21, pp. 778-784, 2003 and then conjugated to an antibody of interest.

[0289] In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics, and analogues thereof, are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Research, vol. 53, pp. 3336-3342, 1993; and Lode et al., Cancer Research, vol. 58, pp 2925-2928, 1998). Calicheamicin has intracellular sites of action but, in certain instances, does not readily cross the plasma membrane. Therefore, cellular uptake through antibody-mediated internalization may, in some embodiments, greatly enhance their cytotoxic effects. Nonlimiting exemplary methods of preparing antibody -drug conjugates with a calicheamicin drug moiety are descnbed, for example, in U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,714,586; U.S. Pat. No. 5,739,116; and U.S. Pat. No. 5,767,285.

[0290] In some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, PDB dimers recognize and bind to specific DNA sequences. The natural product anthramycin, a PBD, was first reported in 1965 (Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp 5793-5795, 1965; and Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp. 5791-5793, 1965). Since then, a number of PBDs, both naturally occurring and analogues, have been reported (Thurston et al., Chem. Rev. vol. 1994, pp. 433-465 1994, including dimers of the tricyclic PBD scaffold (U.S Pat. No. 6,884,799; U.S. Pat. No. 7,049,311; U.S. Pat. No. 7,067,511; U.S. Pat. No. 7,265,105; U.S. Pat. No. 7,511,032; U.S. Pat. No. 7,528,126; U.S. Pat. No. 7,557,099). Without intending tobe bound by any particular theory, it is believed that the dimer structure imparts the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer- Verlag, New York, pp. 3-11 (1975); and Hurley and Needham-VanDevanter, Acc. Chem. Res., vol. 19, pp. 230-237, 1986). Dimeric PBD compounds bearing C2 aryl substituents have been shown to be useful as cytotoxic agents (Hartley et al Cancer Res., vol. 70, pp. 6849-6858, 2010; Antonow, J. Med. Chem.\dV 53, pp. 2927-2941, 2010; and Howard et al., Bioorganic andMed. Chem. Letters, vol. 19, pp. 6463-6466, 2009).

[0291] Nonlimiting exemplary PBD dimer components of ADCs are of Formula A:and salts and solvates thereof, wherein: the wavy line indicates the covalent attachment site to the linker; the dotted lines indicate the optional presence of a double bond between Cl and C2 or C2 and C3;R2is independently selected from H, OH, =0, =CH2, CN, R, OR, =CH — RD, =C(RD)2,O — SO2 — R, CO2R and COR, and optionally is further selected from halo or dihalo, wherein RDis independently selected from R, CO2R, COR, CHO, CO2H, and halo;R6and R9are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, MesSn and halo;R7is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, MesSn and halo;Q is independently selected from O, S and NH;R11is either H, or R or, where Q is 0, SO3M, where M is a metal cation;R and R' are each independently selected from optionally substituted Ci-s alkyl, Ci-12 alkyl, C3-8 heterocyclyl, C3-20 heterocycle, and C 5-20 aryl groups, and optionally in relation to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7 -membered heterocyclic nng;R12, R16, R19and R17are as defined for R2, R6, R9and R7, respectively;R" is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. 0, S, N(H), NMe and / or aromatic rings, e.g. benzene or pyridine, which rings are optionally substituted; andX and X' are independently selected from 0, S and N(H).

[0292] In some embodiments, R and R' are each independently selected from optionally substituted C1-12 alkyl, C3-20 heterocycle, and C5-20 aryl groups, and optionally in relation to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7 -membered heterocyclic nng. In some embodiments, R9and R19are H. In some embodiments, R6and R16are H.

[0293] In some embodiments, R7are R17are both 0R7A, where R7Ais an optionally substituted Ci- 4 alkyl. In some embodiments, R7Ais Me. In some embodiments, R7Ais ClwPh. where Ph is a phenyl group. In some embodiments, X is 0. In some embodiments, R11is H. In some embodiments, there is a double bond between C2 and C3 in each monomer unit.

[0294] In some embodiments, R2and R12are independently selected from H and R. In some embodiments, R2and R12are independently R. In some embodiments, R2and R12are independently optionally substituted C5-20 aryl or C5-7 aryl or Cs-io aryl. In some embodiments, R2and R12are independently optionally substituted phenyl, thienyl, napthyl, pyridyl, quinolmyl, onsoquinohnyl. In some embodiments, R2and R12are independently selected from =0, =CH2, =CH — RD, and =C(RD)2. In some embodiments, R2and R12are each =CH2. In some embodiments, R2and R12are each H. In some embodiments, R2and R12are each =0. In some embodiments, R2and R12are each =CF2. In some embodiments, R2and / or R12are independently =C(RD)2. In some embodiments, R2and / or R12are independently =CH — RD.

[0295] In some embodiments, when R2and / or R12is =CH — RD, each group may independently have either configuration shown below:In some embodiments, a =CH — RDis in configuration (I). In some embodiments, R" is a C3 alkylene group or a C5 alkylene group.

[0296] The linkers of PBD dimer-val-cit-PAB-Ab and the PBD dimer-Phe-Lys-PAB-Ab are protease cleavable, while the linker of PBD dimer-maleimide-acetal is acid-labile.

[0297] PBD dimers and ADCs comprising PBD dimers may be prepared according to methods known in the art. See, e.g, WO 2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US 2011 / 0256157; and WO 2011 / 130598.

[0298] In some embodiments, an ADC may comprise anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. While not intending to be bound by any particular theory, studies have indicated that anthracyclines may operate to kill cells by a number of different mechanisms, including: 1) intercalation of the drug molecules into the DNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis; 2) production by the drug of free radicals which then react with cellular macromolecules to cause damage to the cells, and / or 3) interactions of the drug molecules with the cell membrane (see, e.g., C. Peterson et al., “Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia” in Anthracycline Antibiotics In Cancer Therapy; N. R. Bachur, “Free Radical Damage” id. at pp. 97-102). Because of their cytotoxic potential anthracyclines have been used in the treatment of numerous cancers such as leukemia, breast carcinoma, lung carcinoma, ovanan adenocarcinoma and sarcomas (see e.g., P H-Wiemik, m Anthracycline: Current Status and New Developments p 11).

[0299] Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and derivatives thereof. Immunoconjugates and prodrugs of daunorubicin and doxorubicin have been prepared and studied (Kratz et al., Current Med. Chem. , vol. 13, pp. 477-523, 2006; Jeffrey et al., Bioorganic &Med. Chem. Letters, vol. 16, pp. 358-362. 1996; Torgov et al., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc. Natl. Acad. Sci. USA, vol. 97, pp. 829-834, 2000; Dubowchik et al., Bioorg. &.Med. Chem. Letters, vol. 12, pp. 1529-1532, 2002; King et al., J. Med. Chem., vol 45, pp. 4336-4343, 2002; EP 0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugate BR96-doxorubicin reacts specifically with the tumor-associated antigen Lewis-Y and has been evaluated in phase I and II studies (Saleh et al., J. Clin. Oncology, vol. 18, pp. 2282-2292, 2000; Ajani et al., Cancer Jour., vol. 6, pp. 78-81, 2000; Tolcher et al., J. Clin. Oncology, vol. 17, pp. 478-484, 1999).

[0300] PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clinical Cancer Research, vol. 11, pp. 1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin and has been under clinical evaluation (Grandi et al. Cancer Treat. Rev. vol.17, pp. 133-138, 1990; Ripamonti et al. Brit. J. Cancer, vol. 65, pp. 703-707, 1992), including phase II / III trials for hepatocellular carcinoma (Sun et al., Proceedings of the American Society for Clinical Oncology, vol. 22, Absl448, 2003; Quintieri, Proceedings of the American Association of Cancer Research, vol. 44:1st Ed, Abs 4649, 2003; and Pacciarini et al., Jour. Clin. Oncology, vol. 24, p. 14116, 2006).

[0301] Anthracyclines, including PNU-159682, may be conjugated to antibodies through several linkage sites and a vanety of linkers (US 2011 / 0076287; W02009 / 099741; US 2010 / 0034837; WO 2010 / 009124), including the linkers described herein.

[0302] Other anti-cancer agents also include geldanamycin (Mandler et al., J. Nat. Cancer Inst., vol. 92, pp. 1573-1581, 2000; Mandler et al., Bioorganic &.Med. Chem. Letters, vol. 10, pp. 1025- 1028, 2000; Mandler et al., Bioconjugate Chem., vol. 13, pp. 786-791, 2002); and enzymatically active toxins and fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, e.g., WO 93 / 21232.

[0303] Anti-cancer agents also include compounds with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease).

[0304] In certain embodiments, an immunoconjugate may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212and radioactive isotopes of Lu. In some embodiments, when an immunoconjugate is used for detection, it may compnse a radioactive atom for scintigraphic studies, for example Tc" or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen-15, oxygen- 17, gadolinium, manganese or iron. Zirconium-89 may be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0305] The radio- or other labels may be incorporated in the immunoconjugate in known ways. For example, a peptide may be biosynthesized or chemically synthesized using suitable amino acid precursors comprising, for example, one or more fluorine-19 atoms in place of one or more hydrogens. In some embodiments, labels such as Tc", I123, Re186, Re188and In111can be attached via a cysteine residue in the antibody. In some embodiments, yttrium-90 can be attached via a lysine residue of the antibody. In some embodiments, the lodogen method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978) can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes certain other methods.Drug Loading

[0306] Drug loading refers to the average number of anti-cancer drug moieties per antibody in an ADC Drug loading may range from 1 to 20 anti-cancer drug moieties (D) per antibody. ADCs of the present disclosure include collections of antibodies conjugated with a range of drug moieties, e.g. from 1 to 20. The average number of drug moieties per antibody used in the preparation of ADCs from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of ADCs in terms of anticancer drag moieties may also be determined. In some instances, separation, purification, and characterization of homogeneous ADCs where the number of anti-cancer drug moieties is a certain value from ADCs with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.

[0307] For some antibody-drug conjugates, the number of anti-cancer drug moieties may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. In certain embodiments, higher drag loading, e.g. >5, may cause aggregation, insolubility, toxicity, or loss ofcellular permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an ADC range from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8 and may be about 2 to about 5 (U.S. Pat. No. 7,498,298).

[0308] In certain embodiments, fewer than the theoretical maximum of anti-cancer drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed herein. Generally, antibodies do not contain many free and reactive cysteine thiol groups which may be linked to an anti-cancer drug moiety. Indeed, most cysteine thiol residues in antibodies exist as disulfide bridges. In certain embodiments, an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.

[0309] The loading (drug / antibody ratio) of an ADC may be controlled in different ways, and for example, by: (i) limiting the molar excess of drug- linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (lii) partial or limiting reductive conditions for cysteine thiol modification.

[0310] It is to be understood that where more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, then the resulting product is a mixture of ADCs with a distribution of one or more anti-cancer drug moieties attached to an antibody. The average number of drug moieties per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug. Individual ADCs may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography (see, e.g., McDonagh et al., Prot. Engr. Design & Selection, vol. 19, pp. 299-307, 2006; Hamblett et al., Clin Cancer Res., vol. 10, pp. 7063-7070, 2004). In certain embodiments, a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.Pharmaceutical Formulations

[0311] In each of the embodiments of the treatment methods described herein, the conditionally active anti AXL ADCs may be delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the immunoconjugate is administered to a subj ect in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder. Thus, an aspect of the disclosure relates to a method for treating a disease, and in aparticular embodiment adenoid cystic carcinoma, comprising administering to a subject in need thereof a therapeutically effective amount of an ADC of the disclosure.

[0312] For administration, the conditionally active anti-AXL immunoconjugate may be formulated as a pharmaceutical composition. The pharmaceutical composition including the conditionally active anti-AXL antibody-drug conjugate can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution or composition containing a pharmaceutically acceptable earner.

[0313] A pharmaceutically acceptable carrier is a material that can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier Other suitable pharmaceutically acceptable carriers are well-known to those in the art. (See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)) Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, and albumin to prevent protein loss on vial surfaces, etc.

[0314] The form of the pharmaceutical compositions, the route of administration, the dosage and the dosage regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. These considerations can be taken into account by a skilled person to formulate suitable pharmaceutical compositions. The pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

[0315] Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be, for example, isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition of, for example, sterilized water or physiological saline, permit the constitution of injectable solutions.

[0316] In some embodiments, tonicity agents, sometimes known as “stabilizers” are present to adjust or maintain the tonicity of a liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter- and intra-molecular interactions. Tonicity agents can be present in any amount of from 0. 1% to 25% by weight, preferably 1 to 5% by weight, based on the total weight of the pharmaceutical composition. Typical tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0317] Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients may include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, nbose, ribitol, myoimsitose, myoimsitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0318] Non-ionic surfactants or detergents (also known as “wetting agents”) may be employed to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation- induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants may be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, for example about 0.07 mg / ml to about 0.2 mg / ml.

[0319] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poly oxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc ), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty' acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chlorides such as benzethonium chloride.

[0320] The doses used for administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. To prepare pharmaceutical compositions, an effective amount of the antibody or antibody fragment may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0321] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringcabilily exists. Thepharmaceutical form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0322] The carrier can also be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0323] Sterile injectable solutions are prepared by incorporating the active compounds in the required amounts in the appropriate solvent with one or more of the other ingredients enumerated above, as may be required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fi Itered solution thereof

[0324] The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of dimethyl sulfoxide (DMSO) as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.

[0325] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein

[0326] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Somevariation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0327] Pharmaceutical formulations containing conditionally active anti-AXL antibody drug conjugates as described herein are prepared by mixing such antibody or antibody fragment having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition. Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0328] Exemplary' pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral -active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitmases.

[0329] Exemplary' lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and W02006 / 044908, the latter formulations including a histidme-acetate buffer.Therapeutic Methods

[0330] Any of the conditionally active anti-AXL immunoconjugates provided herein may be used in therapeutic methods. In one aspect, a conditionally active anti-AXL immunoconjugate for use as a medicament is provided. In further aspects, a conditionally active anti-AXL immunoconjugatefor use in treating cancer and in particular adenoid cystic carcinoma is provided. In certain embodiments, a conditionally active anti-AXL antibody immunoconjugate for use in a method of treatment is provided. In certain embodiments, the disclosure provides a conditionally active anti- AXL immunoconjugate for use in a method of treating an individual having adenoid cystic carcinoma comprising administering to the individual an effective amount of the conditionally active anti-AXL immunoconjugate.

[0331] In a further aspect, the disclosure provides for the use of a conditionally active anti-AXL immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of adenoid cystic carcinoma. In a further embodiment, the medicament is for use in a method of treating adenoid cystic carcinoma comprising administering to an individual having adenoid cystic carcinoma an effective amount of the medicament.

[0332] In one embodiment, the methods of treating adenoid cystic carcinoma comprise administering an immunoconjugate that includes the conditionally active anti-AXL antibody or antibody fragments of the disclosure, conjugated to an agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent, and a cytotoxic agent. The immunoconjugate is an antibody-drug conjugate (ADC) in which a Conditionally Active Biologic (CAB) anti-AXL antibody is conjugated to one or more drug moiety via a cleavable linker (CAB-AXL-ADC). For example, the CAB-AXL-ADC is mAbBA3011-cleavable linker-MMAE(n), in which the dmg moiety is monomethyl auristatin E (MMAE), and (n) is an integer between 1 and 4, inclusive, where mAbBA3011 comprises the anti-AXL antibody encoded by the nucleic acid sequences of SEQ ID NOS. 9 and 12 and having a heavy chain amino acid sequence of SEQ ID NO: 128 and a light chain amino acid sequence of SEQ ID NO: 129. The cleavable linker may be any of those provided herein. In one embodiment the cleavable linker is mc-vc-PAB. In another embodiment the cleavable linker is SPC-BA-007. In still another embodiment the cleavable linker is SPC-BA-019. In yet another embodiment the cleavable linker is SPC-BA-017.

[0333] In some embodiments is provided a therapeutic regimen in which the CAB-AXL-ADC of the present disclosure is administered to a subject at a dosage of from 0.3 mg / kg to 2.0 mg / kg at an interval of once or twice every 2 weeks. In certain embodiments, the CAB-AXL-ADC is administered at a dose of 1.8 mg / kg once or twice every 2 weeks. In one particular embodiment, the CAB-AXL-ADC is administered at a dose of 1.8 mg / kg once every 2 weeks (Q2W). In certain therapeutic regimens the CAB-AXL-ADC is mAbBASO 11 -(cleavable linker-auristatin)nwhere n is a number from 1 to 10 inclusive. In some embodiments, n is a number from 1 to 6, while in still other embodiments n is a number from 1 -4. In other embodiments the aunstatin is monomethyl auristatin F (MMAF), while in other embodiments the auristatin is monomethyl auristatin E(MMAE). In one particular therapeutic regimen, a subject having adenoid cystic carcinoma is administered mAbBA-3011-(mc-vc-PAB-MMAE)4 at a dose of 1.8 mg / kg once every 2 weeks.

[0334] In some embodiments, a granulocyte colony stimulating factor such as filgrastim, PEG- filgrastim or a biosimilar is administered in accordance with the applicable label insert as part of the therapeutic regimen. In certain embodiments, the granulocyte colony stimulating factor is administered 48 to 72 hours after the CAB-AXL-ADC is administered. In some embodiments the granulocyte colony stimulating factor is administered only after the first dose of the CAB-AXL- ADC, while in other embodiments the granulocyte colony stimulating factor is administered as needed throughout the course of treatment.

[0335] Also provided is a pharmaceutical composition for use as a medicament for the treatment of adenoid cystic carcinoma wherein the pharmaceutical composition comprises the CAB-AXL- ADC of the present disclosure and a pharmaceutically acceptable carrier to be administered at a dose of from 0.3 mg / kg to 2.0 mg / kg at an interval of once or twice every 2 weeks. In certain embodiments, the pharmaceutical composition comprising mAbBA-3011-(mc-vc-PAB-MMAE)4 is administered at a dose from 0.3 mg / kg to 2.0 mg / kg at an interval of once or twice every 2 weeks. In certain embodiments, the CAB-AXL-ADC of the pharmaceutical composition is administered at a dose of 1.8 mg / kg once or twice every 2 weeks. In one particular embodiment, the CAB-AXL- ADC of the pharmaceutical composition is administered at a dose of 1.8 mg / kg once every 2 weeks (Q2W). In certain embodiments the CAB-AXL-ADC of the pharmaceutical composition is mAbBA3011-(cleavable lmker-auristatin)nwhere n is a number from 1 to 10 inclusive. In some embodiments, n is a number from 1 to 6, while in still other embodiments n is a number from 1-4 In other embodiments the auristatin is monomethyl auristatin F (MMAF), while in other embodiments the auristatin is monomethyl auristatin E (MMAE). One particular embodiment provides a pharmaceutical composition for use as a medicament in a subject having adenoid cystic carcinoma at a dose of 1.8 mg / kg once every 2 weeks, said pharmaceutical composition comprising mAbBA-3011-(mc-vc-PAB-MMAE)4 and a pharmaceutical acceptable carrier.

[0336] In yet another embodiment, the pharmaceutically acceptable carrier has a pH of 6.0 and comprises 20 mM histidine-HCl, 70 mg / mL sucrose and 0.5 mg / mL polysorbate 80.

[0337] The conditionally active anti-AXL antibodies or antibody fragments and their immunoconjugates may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the type of adenoid cystic carcinoma (ACC) being treated, whether the ACC being treated is localized or has undergone metastases, the particular mammal being treated, the clinical condition of the individual patient, the site of delivery of the agent, the method of administration, the scheduling of administration,and other factors known to medical practitioners. In one embodiment, the subject suffers from Type I ACC. In another embodiment, the subject suffers from Type II ACC.

[0338] In certain embodiments, the conditionally active anti-AXL immunoconjugates of the present disclosure are administered to a patient in order to cause regression of the ACC tumor or tumors. In other instances, the conditionally active anti-AXL immunoconjugates disclosed herein are administered to a patient with ACC to prevent further progression of the disease. The duration of treatment will depend on the type of ACC present (Type I or Type II), the extent of the disease, the result to be achieved and the tolerance of the patent to the treatment. Determining the appropriate duration of treatment is well within the skill of a trained medical practitioner.

[0339] The conditionally active anti-AXL immunoconjugates described herein can be administered in combination with other therapies used in cancer treatment. Such treatments include but are not limited to surgical resection, radiation therapy and chemotherapy. Radiation therapy (e.g., radiotherapy, X-ray therapy, irradiation) involves the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered either externally via external beam radiotherapy (EBRT) or internally via brachytherapy. In addition, the treating physician may optionally provide additional treatments to lessen discomfort or limit side-effects associated with the treatment. These include, but are not limited to, pain medication such as morphine and oxycodone, anti-emetics such as ondansetron and aprepitant.Plasma Membrane Scoring of AXL in Tumor (Tumor Membrane P Score)

[0340] In some embodiments, the level of AXL expression in tumor cells is determined prior to treatment. It has been reported that AXL expression is significantly increased in Type II ACC (Ferrarotto et al., Clin. Cancer Res., vol. 27, pp. 852-864, 2021). It is to be understood, however, that determination of AXL expression is optional and not a requirement for treatment of adenoid cystic carcinoma using the conditionally active anti-AXL immunoconjugates disclosed herein.

[0341] AXL is reactive in a subset of tumor cells and macrophages. In tumor cells, AXL is primarily localized to the plasma membrane but can also be observed in the cytoplasm Macrophages that express AXL are often present among tumor cells / nests and within the stroma adjacent to the tumor (tumor-associated stroma or tumor-stroma). AXL macrophage staining is localized to the plasma membrane or the cytoplasm, although not all macrophages label with AXL

[0342] CD68 is expressed in the cytoplasm of macrophages and is a standard biomarker for identification of this immune cell type. Macrophages can be present throughout tissue samples but are often of most interest when present among tumor cells (within the tumor mass) and at the tumor / stroma interface (tumor-associated stroma).

[0343] Because AXL is expressed in both tumor cells and macrophages, a scoring approach to compare AXL and CD68 staining in serial sections of each sample can optionally be used. In this way, the CD68 biomarker is used to identify macrophages in the tumors stained for AXL. That is, the CD68 serial section is used to distinguish AXL reactivity in tumor cells versus macrophages. Using this approach, AXL plasma membrane staining is scored only in tumor cells. CD68 staining is “subtracted out” of the assessment for AXL to provide AXL tumor scoring exclusive of macrophages (hereinafter “tumor membrane P score”).

[0344] The approaches used for scoring AXL and CD68 may be detected by methods, including but not limited to, immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) tumor samples as described below. All samples are also stained with hematoxylin and eosin (H&E) for morphological assessment to assist in scoring.

[0345] AXL plasma membrane expression in tumor is scored semi-quantitatively. The main components of scoring are percentages of cells staining at appropriate differential intensities.

[0346] Percentage scores are assigned to describe the penetrance of plasma membrane staining per sample. Percentages are estimated and reported as an increment, including but not limited to, one of the following increments: 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%. In certain embodiments, the AXL-expressing tumor has a tumor membrane P score of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0347] Differential intensities for plasma membrane staining are recorded using a four-point scale semi-quantitatively (0, 1+, 2+, 3+). On this scale, 0 = null, negative, or non-specific staining, 1+ = low or weak staining, 2+ = medium or moderate staining, and 3+ = high or strong staining.

[0348] When assessing AXL reactivity only in tumor cells (AXL tumor staining exclusive of macrophages), stained slides for both AXL and for CD68 (macrophage biomarker) are required in series. Staining with CD68 is closely compared to staining with AXL in regions of a tumor. Cells that stain with both AXL and CD68 are considered AXL-reactive macrophages (not tumor cells) and are excluded from the AXL score.

[0349] AXL staining in macrophages can make it difficult to score plasma membrane staining in tumor cells when there is a mixed population of positive cell types. To gain a full understanding of AXL expression on the plasma membrane in tumors exclusive of macrophages across cancer indication, both standard Percent Score and H-Score approaches may be used to capture the pattern of reactivity observed.Percent Score Method

[0350] Percent Scores are calculated by summing the percentages of intensities at either >1+, >2+ or >3+ Thus, scores range from 0 to 100.Percent Score >1+ = (% at 1+) + (% at 2+) + (% at 3+)Percent Score >2+ = (% at 2+) + (% at 3+)Percent Score >3+ = (% at 3+).H-Score Method

[0351] The H-Score is calculated by summing the percentage of cells with intensity of expression (brown staining) multiplied by their corresponding differential intensity on a four-point semi quantitative scale (0, 1+, 2+, 3+). Thus, scores range from 0 to 300.H-Score = [ (% at <1) x 0 ] + [ (% at 1+) x 1 ] + [ (% at 2+) x 2 ] + [ (% at 3+) x 3]Scoring of Macrophage Staining

[0352] As stated, CD68 is a standard biomarker for macrophages and AXL can be expressed in this cell type. Slides stained with CD68 and AXL are used to assess the relative abundance of CD68-positive and AXL-positive macrophages in each tumor tissue. Macrophage estimates are made for CD68 and AXL positivity in the following regions: within the tumor mass (called “tumor”) and within tumor-associated stroma or stroma that interacts with tumor (called “tumorstroma”). Tumor-stroma represent the parenchymal response to a tumor. It is the stromal response, outside or adjacent to the outer edge or “face” of the tumor mass. In a tumor, scoring represents the percentage (0-100%) of cells in the tumor mass or tumor nests that are comprised of CD68 or AXL-positive macrophages. In tumor-stroma, macrophage abundance for AXL and CD68 is scored using a semi -quantitative scale of from 0-3. On this scale, 0 represents no positive macrophages, 1 indicates low density of positive macrophages, 2 indicates moderate density of positive macrophages, and 3 indicates high density of positive macrophages.EXAMPLESExample 1. pH-Dependent Binding Affinity of Anti-AXL Antibodies

[0353] Some of the anti-AXL antibodies of the present disclosure were tested in buffers at different pH levels. One type of buffer was a KREBS buffer with 1% bovine semm albumin (BSA) present. The KREBS buffer was titrated to have a pH in the range of 5-7.4. The binding affinity of the antibodies with AXL was measured using an ELISA assay (OD450) and the results are presented in FIG. 2. The two control antibodies (BAP063-3831 and BAP063-3818) were not conditionally active as they have a binding affinity that was not significantly affected by the changes in pH. On the other hand, the anti-AXL antibodies of the present invention are conditionally active since their binding affinity with AXL was dependent on the pH (FIG. 2).

[0354] Table 3 presents the SEQ ID NOs of the antibodies tested and shown in FIG. 2. The antibody BAP063-4007 in Fig. 2 is encoded by a combination of SEQ ID NOS. 9 and 12 and have heavy and light chain variable region amino acid sequences of SEQ ID NO:128 and SEQ ID NO: 129 respectively. BAP063-4007 is also identified as mAbBA3011.Table 3: Anti-AXL antibodiesExample 2: Clinical EvaluationDrug Product

[0355] BA3011 Drug Product was supplied as a sterile frozen liquid or a lyophilized dosage form. The composition of the drug product is shown in Table 4. The composition of the drug product was 10.0 mg / mL of BA3011 drug substance in 20 mM histidine buffer, pH 6.0 with 70 mg / mL sucrose and 0.5 mg / mL polysorbate 80.Table 4: Composition of Drug ProductPatients

[0356] Eligible patients in the study were 18 years and older and were required to have a histologically or cytologically confirmed locally advanced unresectable or metastatic solid ACC tumor. Patients must have failed all available standard of care therapies and either have no curative therapy options or not be eligible for, intolerant of, or refuse standard therapy. In addition, patients had measurable disease per the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and adequate organ function. No predefined expression level of AXL was required for patients.Study Treatment

[0357] Mecbotamab vedotin (mAbBA-3011-(mc-vc-PAB-MMAE)4) was administered as an IV infusion of 1.8 mg / kg of the patient weight every 2 weeks until disease progression, unacceptable toxicity, or another reason for treatment discontinuation.Assessments

[0358] The safety of mecbotamab vedotin was assessed by monitoring adverse events (AEs), including dose-limiting toxicities, which were coded by system organ class and preferred term using the Medical Dictionary for Regulatory Affairs (MedDRA), and graded according to NCI CTCAE version 4.03 / 5. Evaluation of safety also included clinical laboratory tests results and physical exam findings. Anti-tumor activity was assessed using radiographic tumor assessments, performed at baseline and approximately every 6 weeks until 12 weeks, then every 8 weeks for 1 year, and then every 12 weeks thereafter using computed tomography (CT). Evaluation of radiographic scans was performed by both the investigator and by Independent Central Review, at the discretion of the Sponsor.Statistical Analyses

[0359] All safety parameters were summarized based on the As-Treated Population, which included all ACC patients who received any amount of mecbotamab vedotin.

[0360] All efficacy analyses were performed using the Response-Evaluable Population, which included all patients in the As-Treated Population who had a baseline disease assessment and the potential to be followed for at least 12 weeks at the time of the data cutoff and either had at least 1 post-baseline disease assessment and / or discontinued treatment due to death or disease progression. Efficacy endpoints included assessment of objective response per RECIST vl. 1 using investigator assessment. The objective response rate (ORR) was defined as the proportion of patients with a best overall response of confirmed complete response (CR) or confirmed partialresponse (PR) that occurred prior to the initiation of subsequent anticancer treatment. Objective response rate was estimated with a 95% confidence interval (CI) using the exact probability method. Other efficacy endpoints included duration of response (DOR), progression-free survival (PFS), best overall response (BOR), disease control rate (DCR), time to response (TTR), overall survival (OS), and percent change from baseline in target lesion sum of diameters. All secondary efficacy endpoints except OS were based on RECIST vl.l using investigators’ assessment. Response rates and their 95% CI were estimated using the exact probability method. Time-to-event data were summarized using Kaplan-Meier estimates including median and hazard ratio (HR) with their Cis.Safety

[0361] The median number of mecbotamab vedotin infusions was 12.5 (range, 2-36), equating to a median treatment duration of 198 days (range, 27 to 541).

[0362] All 28 patients experienced at least 1 treatment-emergent adverse event (TEAE), the most common of which were fatigue, peripheral sensory neuropathy, nausea, and increased aspartate aminotransferase.

[0363] Grade 3 or 4 TEAEs were reported for 12 patients (43%), the most common of which were fatigue, anemia, neutrophil count decreased, pneumonia, dysphagia, and sepsis in 2 patients each (7%). Treatment-related Grade 3 / 4 AEs were reported for 6 patients (21%), including preferred terms of anemia and neutrophil count decreased (in 2 patients each, 7%), and neutropenia, sepsis, and fatigue (in 1 patient each, 4%). Eight patients experienced serious adverse events (SAEs). These were most frequently (4 patients) events associated with pneumonia and associated causes (e g., aspiration and dysphagia) or symptoms (e.g., pleural effusion). In each case, these were assessed as unrelated to mecbotamab vedotin by the investigator and instead were found to be due to the underlying disease. One other patient experienced SAEs assessed by the investigator as unrelated to mecbotamab vedotin; events of malaise and fall that were also considered to be due to the patient’s underlying disease. SAEs in 3 patients were considered at least possibly related to mecbotamab vedotin: One patient each wi th infusion site extravasation, anemia, and neutropema / septic shock. The events of infusion site extravasation and anemia resolved without sequelae; however, the patient with neutropenia and septic shock died due to these events. Four patients (14%) discontinued treatment due to TEAEs, including 2 patients who discontinued due to peripheral neuropathy, 1 patient who discontinued due to fatigue, and the patient who died from septic shock, discussed above.Efficacy

[0364] The best overall response for the “as treated” population was a partial response (PR) for 1 patient (4%) and stable disease (SD) for 25 patients (89%), equating to a disease control rate (DCR) of 93%. Response evaluation data were not available for 1 patient (due to death) and 1 patient had progressive disease. The median duration of SD was 10.6 months (95% CI: 8.4, 13.1), range 1.6 to 18.4 months. The median progression-free survival (PFS) was 11.9 months (95% CI: 8.4, 12.7).

[0365] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially o”' are also provided for.

Claims

WHAT IS CLAIMED IS:

1. A method of treating adenoid cystic carcinoma, comprising a step of administering to a subject having adenoid cystic carcinoma a pharmaceutical composition comprising AbCAB- (linker-D)n and a pharmaceutically acceptable carrier at a dose of 1.8 mg / kg once every two weeks, wherein:AbCAB is a conditionally active anti-AXL antibody or antigen binding antibody fragment thereof, linker is a cleavable linker,D is an anti-cancer agent, and n is an integer from 1 to 4.

2. The method of claim 1, wherein the conditionally active anti-AXL antibody or antigen binding antibody fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from amino acid sequences of SEQ ID NOs: 129, 131, 133, 135, 136, and 137; and a light chain variable region comprising an amino acid sequence selected from amino acid sequences of SEQ ID NOs: 48, 128, 132, 134, and 138.

3. The method of claim 1 or 2, wherein the conditionally active anti-AXL antibody or antigen-binding antibody fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 128 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 129; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:131 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 132; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 133 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 134; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 135 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 128; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 136 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:48; and a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 137 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 138.

4. The method of claim 1, wherein the conditionally active anti-AXL antibody or antigenbinding antibody fragment thereof compnses a heavy chain variable region comprising an aminoacid sequence of SEQ ID NO: 128 and a light chain variable region compnsing an amino acid sequence of SEQ ID NO: 129.

5. The method of claim 1, wherein the conditionally active anti-AXL antibody or antigenbinding antibody fragment thereof compnses a heavy chain variable region encoded by SEQ ID NO: 12 and a light chain variable region encoded by SEQ ID NO: 9.

6. The method of any one of claims 1-5, wherein the conditionally active anti-AXL antibody or antigen binding antibody fragment thereof compnses a heavy chain variable region comprising: a CDR-H1 comprising an amino acid sequence of WGATMN (SEQ ID NO: 123); a CDR-H2 comprising an amino acid sequence of LIKPSNGGTSYNQKFKG (SEQ ID NO:118); and a CDR-H3 comprising an amino acid sequence of GHYESYEAMDYWG (SEQ ID NO: 124); and a light chain variable region comprising: a CDR-L1 comprising an amino acid sequence of KA.SQDVVSAVA (SEQ ID NO: 125); a CDR-L2 comprising an amino acid sequence of WQDTRHT (SEQ ID NO: 126); and a CDR-L3 comprising an amino acid sequence of QEHFSPPLT (SEQ ID NO: 127).

7. The method of any one of claims 1-6, wherein the pharmaceutical composition is administered by intravenous infusion.

8. The method of any one of claims 1-7, wherein the linker is9. The method of any one of claims 1-7, wherein the linker is10. The method of any one of claims 1-7, wherein the linker is11. The method of any one of claims 1-7, wherein the linker is12. The method of any one of claims 1-11, wherein the pharmaceutically acceptable carrier has a pH of 6.0 and comprises 20 mM histidine-HCl, 70 mg / mL sucrose and 0.5 mg / mL polysorbate 80.

13. The method of any one of claims 1-12, wherein n is 4.

14. The method of any one of claims 1-13, wherein the anti-cancer agent is an auristatin.

15. The method of claim 14, wherein the auristatin is monomethyl auristatin E.

16. The method of any one of claims 1-15, wherein the adenoid cystic carcinoma is a type II adenoid cystic carcinoma.

17. The method of any one of claims 1-15, wherein the adenoid cystic carcinoma is a type I adenoid cystic carcinoma.

18. A pharmaceutical composition for use as a medicament in a treatment of adenoid cystic carcinoma wherein the pharmaceutical composition composes AbCAB-(lmker-D)nand a pharmaceutically acceptable carrier for administration of the pharmaceutical composition at a dose of 1.8 mg / kg pf patient body weight once every 2 weeks, whereinAbCAB is a conditionally active anti-AXL antibody or antigen binding antibody fragment thereof, linker is a cleavable linker,D is an anti-cancer agent, and n is an integer from 1 to 4.

19. The pharmaceutical composition of claim 18, wherein the conditionally active anti-AXL antibody or antigen binding antibody fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from amino acid sequences of SEQ ID NOs:129, 131, 133, 135, 136, and 137; and a light chain variable region comprising an amino acid sequence selected from amino acid sequences of SEQ ID NO 48, 128, 132, 134, and 138.

20. The pharmaceutical composition of claim 18, wherein the conditionally active anti-AXL antibody or antigen-binding antibody fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 128 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 129; aheavy chain variable region comprising an amino acid sequence of SEQ ID NO:131 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 132; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 133 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 134; a heavy chain variable region comprising an ammo acid sequence of SEQ ID NO: 135 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 128; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 136 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:48; and a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 137 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 138.

21. The pharmaceutical composition of claim 18, wherein the conditionally active anti-AXL antibody or antigen-binding antibody fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 128 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 129.

22. The pharmaceutical composition of claim 18, wherein the conditionally active anti-AXL antibody or antigen-binding antibody fragment thereof comprises a heavy chain vanable region encoded by SEQ ID NO: 12 and a light chain variable region encoded by SEQ ID NO: 9.

23. The pharmaceutical composition of any one of claims 18 to 22, wherein the conditionally active anti-AXL antibody or antigen binding antibody fragment thereof comprises a heavy chain variable region comprising: a CDR-H1 comprising an amino acid sequence of WGATMN (SEQ ID NO: 123); a CDR-H2 comprising an amino acid sequence of LIKPSNGGTSYNQKFKG (SEQ ID NO:118); and a CDR-L3 comprising an amino acid sequence of GHYESYEAMDYWG (SEQ ID NO: 124); and a light chain variable region comprising: a CDR-L1 comprising an amino acid sequence of KASQDVVSAVA (SEQ ID NO: 125); a CDR-L2 comprising an amino acid sequence of WQDTRHT (SEQ ID NO: 126); and a CDR-L2 comprising an amino acid sequence of QEHFSPPLT (SEQ ID NO: 127).

24. The pharmaceutical composition of any one of claims 18 to 23, wherein the pharmaceutical composition is configured for administration by intravenous infusion.

25. The pharmaceutical composition of any one of claims 18-24, wherein the linker is26. The pharmaceutical composition of any one of claims 18-24, wherein the linker is27. The pharmaceutical composition of any one of claims 18-24, wherein the linker is28. The pharmaceutical composition of any one of claims 18-24, wherein the linker is29. The pharmaceutical composition of any one of claims 18-28, wherein the pharmaceutically acceptable carrier has a pH of 6.0 and comprises 20 mM histidine-HCl, 70 mg / mL sucrose and 0.5 mg / mL polysorbate 80.

30. The pharmaceutical composition of any one of claims 18-29, wherein n is 4.

31. The pharmaceutical composition of any one of claims 18-30, wherein the anti-cancer agent is an auristatin.

32. The pharmaceutical composition of claim 31, wherein the auristatin is monomethyl auristatin E.

33. The pharmaceutical composition of any one of claims 18-32, wherein the adenoid cystic carcinoma is a type II adenoid cystic carcinoma.

34. The pharmaceutical composition of any one of claims 18-32, wherein the adenoid cystic carcinoma is a type I adenoid cystic carcinoma.

Images