Pharmaceutical compositions comprising anti-tissue factor antibody-drug conjugates

Abstract

Provided herein is a pharmaceutical composition comprising an anti-tissue factor antibody-drug conjugate (ADC) and a pharma- ceutical acceptable excipient.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 301,994, filed January 21, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a computer readable sequence listing submitted herewith in XML file format, the entire contents of which are incorporated herein by reference. The sequence listing XML file submitted herewith is entitled "14529-102-228_SEQ_LISTING.xml", was created on January 18, 2023, and is 43,585 bytes in size.

[0003] The present disclosure relates to pharmaceutical compositions comprising anti-tissue factor antibody-drug conjugates (ADCs). [Background technology]

[0004] Blood coagulation involves a complex series of processes that result in blood clotting. Tissue factor (TF) plays a key role in these clotting processes. TF is a cell surface receptor for the serine protease factor VIIa (FVIIa). The TF / FVIIa complex catalyzes the conversion of the inactive protease factor X (FX) to the active protease factor Xa (FXa). FXa and its cofactor FVa form the prothrombinase complex, which generates thrombin from prothrombin. Thrombin converts soluble fibrinogen into insoluble chains of fibrin and catalyzes many other coagulation-related processes.

[0005] TF is overexpressed in multiple types of solid tumors, and in cancer, TF / FVIIa signaling can support angiogenesis, tumor progression, and metastasis.

[0006] There is a need in the art for stable formulations comprising anti-tissue factor antibody-drug conjugates (ADCs) useful for treating diseases or disorders such as solid tumors and cancer. The present disclosure addresses such a need. Summary of the Invention

[0007] Provided herein is a pharmaceutical composition comprising an anti-tissue factor antibody-drug conjugate.

[0008] In some embodiments, the present disclosure provides: (i) An antibody-drug conjugate of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, the Ab is a tissue factor (TF) antibody, the Ab comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3; VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 are from the antibody designated 25A3; n is an integer of 1 or more, an antibody-drug conjugate, or a pharma- ceutically acceptable salt thereof, wherein the succinimidyl group is covalently attached to the Ab; (ii) a buffer solution; and (iii) a tonicifier; and (iv) a surfactant.

[0009] In some embodiments, the succinimidyl group is attached to the Ab via a cysteine ​​residue in the Ab.

[0010] In some embodiments, the buffer is histidine, the tonicity agent is sucrose, and the surfactant is polysorbate 80. In some embodiments, the pharmaceutical compositions provided herein comprise an antibody-drug conjugate provided herein, histidine, hydrochloric acid, sucrose, and polysorbate 80.

[0011] In some embodiments, provided herein is a pharmaceutical composition comprising 5-20 mg / mL of an antibody-drug conjugate provided herein, 10-50 mM histidine, 5-10% (w / v) sucrose, and 0.01-0.05% (w / v) polysorbate 80, wherein the pharmaceutical composition has a pH value of 5-6.

[0012] In some embodiments, provided herein is a pharmaceutical composition comprising 10 mg / mL of an antibody-drug conjugate provided herein, 20 mM histidine, 8% (w / v) sucrose, and 0.02% (w / v) polysorbate 80, wherein the pharmaceutical composition has a pH value of about 5.5.

[0013] In another aspect, provided herein is a pharmaceutical composition provided herein for use in treating a disease or disorder in a subject. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition is stored in a glass vial or a polycarbonate bottle.

[0014] In another aspect, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject a pharmaceutical composition provided herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), urothelial carcinoma, ovarian cancer (e.g., epithelial), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., with squamous cell histology), and pancreatic cancer. In some embodiments, the subject is a human subject. [Brief description of the drawings]

[0015] [Figure 1A] The figures show the change in percent monomer, percent high molecular weight (HMW), and percent low molecular weight (LMW) species as measured by SEC for formulations prepared in different buffers and stored at 40° C. / 75% RH for up to 2 weeks. A slight change in molecular size distribution in histidine buffer is shown. In each of these figures, the three bars for each test formulation represent, from left to right, the TO, 1 week, and 2 week storage periods, respectively. [Figure 1B] Figure 1 shows the change in percent monomer, percent high molecular weight (HMW), and percent low molecular weight (LMW) species as measured by SEC for formulations prepared in different buffers and stored at 40°C / 75%RH for up to 2 weeks. A clear change in molecular size distribution in citrate buffer is shown. In each of these figures, the three bars for each test formulation represent, from left to right, the TO, 1 week, and 2 week storage periods, respectively. [Figure 1C] Figure 1 shows the change in percent monomer, percent high molecular weight (HMW), and percent low molecular weight (LMW) species as measured by SEC for formulations prepared in different buffers and stored at 40°C / 75% RH for up to 2 weeks. A clear change in molecular size distribution in phosphate buffer is shown. In each of these figures, the three bars for each test formulation represent, from left to right, the TO, 1 week, and 2 week storage periods, respectively.

[0016] [Figure 2A] Figure 1 shows the statistical significance of percent monomer change during storage for up to 2 weeks at 40° C. as a function of buffer type, concentration (mM), and pH. It shows that formulations prepared with citric acid and histidine had less change in percent monomer than the formulation prepared with phosphoric acid. [Figure 2B]Figure 1 shows the statistical significance of percent monomer change during storage for up to 2 weeks at 40° C. as a function of buffer type, concentration (mM), and pH. It shows that the 30 mM concentration showed less change in percent monomer than the 10 mM and 50 mM concentrations. [Figure 2C] Figure 1 shows the statistical significance of percent monomer change during storage for up to 2 weeks at 40° C. as a function of buffer type, concentration (mM), and pH. It is shown that smaller differences in percent monomer occurred at pH 5 and pH 6. [Figure 2D] Figure 1 shows the statistical significance of percent monomer change during up to two weeks storage at 40° C. as a function of buffer type, concentration (mM), and pH. It shows that formulations prepared with histidine showed less change in percent monomer during up to two weeks storage at 40° C.

[0017] [Diagram 3] Figure 1 shows the change in the main peak percent as analyzed by iCE. Formulations were prepared with histidine and stored at -20°C or -80°C for up to 6 months. At both temperatures, formulation 1, prepared at pH 7 with 50 mM histidine, showed a consistent decrease in the main peak percent. Other formulations prepared with histidine did not show a significant change in the main peak percent.

[0018] [Figure 4] It is shown that all formulations prepared with citric acid showed an increase in the percentage of acidic species during storage up to 2 months at 2° C.-8° C. The four bars for each test formulation, from left to right, represent storage periods of TO, 2 weeks, 1 month, and 2 months, respectively.

[0019] [Diagram 5] It is shown that all formulations prepared with phosphoric acid showed a decrease in the percentage of the main peak during storage up to 2 months at 2° C.-8° C. The four bars for each tested formulation, from left to right, represent storage periods of T0, 2 weeks, 1 month, and 2 months, respectively.

[0020] [Figure 6] The statistical significance of the main peak percent change is shown as a function of buffer concentration (mM) in the formulation during storage at 2° C.-8° C. for up to 2 months.

[0021] [Figure 7] The statistical significance of the main peak percent change is shown as a function of pH in the formulations during storage at 2° C.-8° C. up to 2 months.

[0022] [Figure 8] The statistical significance of the main peak percent change is shown as a function of pH in the formulations up to 2 weeks of storage at 25°C.

[0023] [Figure 9-1] DLS analysis of mean particle size and percent polydispersity in formulations at TO (top) and after storage for up to 6 months at -80° C. or -20° C. (bottom) is shown. No significant changes were observed for the formulations tested, with the exception of formulations 1 and 4. [Figure 9-2] DLS analysis of mean particle size and percent polydispersity in formulations at TO (top) and after storage for up to 6 months at -80° C. or -20° C. (bottom) is shown. No significant changes were observed for the formulations tested, with the exception of formulations 1 and 4.

[0024] definition Unless otherwise defined, notations and other scientific terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a difference from what is commonly understood in the art.

[0025] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Terms such as "including," "etc," and the like are intended to convey an inclusion without limitation unless specifically indicated otherwise.

[0026] The term "about" or "approximately" refers to and encompasses the indicated value and ranges above and below that value. In certain embodiments, the term "about" or "approximately" refers to the specified value ±10%, ±5%, or ±1%. In certain embodiments, where applicable, the term "about" or "approximately" refers to the specified value(s) ±1 standard deviation of that value(s).

[0027] The terms "tissue factor" and "TF" are used interchangeably herein to refer to TF, any variants (e.g., splice variants and allelic variants), isoforms, and species homologs of TF expressed naturally by cells or by cells transfected with the TF gene. In some aspects, the TF protein is a TF protein naturally expressed by a primate (e.g., monkey or human), rodent (e.g., mouse or rat), dog, camel, cat, cow, goat, horse, pig, or sheep. TF is a cell surface receptor for the serine protease factor VIIa. It is often constitutively expressed by certain cells surrounding blood vessels and in some disease conditions. In certain embodiments, the antibodies and antibody-drug conjugates (ADCs) described herein bind to the extracellular domain (SEQ ID NO: 39) of human tissue factor (TF).

[0028] The term "immunoglobulin" generally refers to a class of structurally related proteins that contain two paired polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In "intact immunoglobulins", all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, heavy chains typically contain a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region typically comprises H1 , C H2 , and C H3 Each light chain typically contains three domains, abbreviated as V L ) and a light chain constant region. The light chain constant region typically comprises L It contains one domain, abbreviated as .

[0029] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0030] V H Area and V L The regions can be further subdivided into regions of hypervariability ("hypervariable regions" (HVRs), also called "complementarity determining regions" (CDRs)), interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). Each V H and V Lgenerally contains three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and affect the antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.

[0031] "Complementarity determining regions (CDRs)" are the amino acid sequences of an immunoglobulin (Ig or antibody) V HIt refers to one of the three hypervariable regions (H1, H2, or H3) in the non-framework region of the β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of the antibody VL β-sheet framework. CDRs are variable region sequences flanked within the framework region sequences. CDRs are well recognized in the art and are defined by Kabat, for example, as the most hypervariable regions in the antibody variable (V) domain. See Kabat et al., J Biol Chem, 1977, 252:6609-6616 and Kabat, Adv Protein Chem, 1978, 32:1-75, each of which is incorporated by reference in its entirety. CDRs are also structurally defined by Chothia as those residues that are not part of the conserved β-sheet framework and can adopt different conformations. See Chothia and Lesk, J Mol Biol, 1987, 196:901-917, each of which is incorporated by reference in its entirety. Both Kabat and Chothia nomenclature are well known in the art. AbM, Contact, and IMGT also define CDRs. CDR positions within standard antibody variable domains have been determined by comparison of numerous structures. See Morea et al., Methods, 2000, 20:267-279 and Al-Lazikani et al., J Mol Biol, 1997, 273:927-48, each of which is incorporated by reference in its entirety. Because the number of residues within hypervariable regions varies in different antibodies, additional residues relative to the standard positions are customarily numbered a, b, c, etc. next to the residue number in the standard variable domain numbering scheme (Al-Lazikani et al., supra). Such terminology is well known to those of skill in the art.

[0032] Several hypervariable region descriptive methods are in use and are included herein. Kabat CDRs are based on sequence variability and are the most commonly used. See Kabat et al. (1992) Sequences of Proteins of Immunological Interest, DIANE Publishing: 2719, which is incorporated by reference in its entirety. Chothia instead refers to the location of structural loops (Chothia and Lesk, supra). AbM hypervariable regions are a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. Contact hypervariable regions are based on analysis of available complex crystal structures. Residues from each of these hypervariable regions are set forth in Table 2 provided herein.

[0033] More recently, a universal numbering system, the ImMunoGeneTics (IMGT) Information System™, has been developed and widely adopted. See Lefranc et al., Dev Comp Immunol, 2003, 27:55-77, which is incorporated by reference in its entirety. IMGT is an integrated information system that is specific to human and other vertebrate immunoglobulins (IGs), T cell receptors (TRs), and major histocompatibility complexes (MHCs). IMGT CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and occurs in structures called loops, by using a numbering system that aligns variable domain sequences according to structural features, CDR and framework residues are readily identified. The correspondence between Kabat, Chothia, and IMGT numbering is also well known in the art (Lefranc et al., supra). The Exemplary system presented herein combines the Kabat and Chothia CDR definitions.

[0034] The light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of their constant domain.

[0035] Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0036] The term "subject" refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, pigs, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with an antibody provided herein.

[0037] The term "therapeutically effective amount" or "effective amount" refers to an amount of an antibody or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising an antibody-drug conjugate (ADC) and a pharma- ceutically acceptable excipient. Also provided herein is a pharmaceutical composition comprising a population of ADCs and a pharma- ceutically acceptable excipient. ADCs and populations of ADCs are described in more detail below. In some embodiments, the excipient provided herein can be a buffer, a tonicity agent, a surfactant, or a combination thereof.

[0039] In some embodiments, the pharmaceutical compositions provided herein comprise an antibody-drug conjugate(s) provided herein, a buffer, an isotonicity agent, and a surfactant. In some embodiments, the pharmaceutical compositions provided herein further comprise an agent that adjusts the pH value of the pharmaceutical composition. In some embodiments, the buffer is histidine, the isotonicity agent is sucrose, the surfactant is polysorbate 80, and / or the agent that adjusts the pH value of the pharmaceutical composition is hydrochloric acid.

[0040] In some embodiments, the pharmaceutical compositions provided herein comprise an antibody-drug conjugate(s) provided herein, histidine, hydrochloric acid, sucrose, and polysorbate 80. In some embodiments, the pharmaceutical compositions provided herein consist essentially of an antibody-drug conjugate(s) provided herein, histidine, hydrochloric acid, sucrose, and polysorbate 80.

[0041] In some embodiments, provided herein is a pharmaceutical composition comprising 5-20 mg / mL of an antibody-drug conjugate provided herein, 10-50 mM histidine, 5-10% (w / v) sucrose, and 0.01-0.05% (w / v) polysorbate 80, wherein the pharmaceutical composition has a pH value of 5 to 6. In some embodiments, provided herein is a pharmaceutical composition comprising 5-20 mg / mL of an antibody-drug conjugate provided herein, 10-30 mM histidine, 5-10% (w / v) sucrose, and 0.01-0.05% (w / v) polysorbate 80, wherein the pharmaceutical composition has a pH value of 5 to 6.

[0042] In some embodiments, provided herein is a pharmaceutical composition comprising 10 mg / mL of an antibody-drug conjugate provided herein, 20 mM histidine, 8% (w / v) sucrose, and 0.02% (w / v) polysorbate 80, wherein the pharmaceutical composition has a pH value of about 5.5. In some embodiments, provided herein is a pharmaceutical composition consisting essentially of 10 mg / mL of an antibody-drug conjugate provided herein, 20 mM histidine, 8% (w / v) sucrose, and 0.02% (w / v) polysorbate 80, wherein the pharmaceutical composition has a pH value of about 5.5.

[0043] In some embodiments, the pharmaceutical compositions are lyophilized. The lyophilized powders can be reconstituted for administration into solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels. Lyophilized powders may be prepared by dissolving the ADC provided herein in a suitable solvent. The solvent may contain excipients or other pharmacological components of the powder that improve stability, or a reconstituted solution prepared from the powder. Subsequent sterile filtration of the solution may be performed under standard conditions known to those of skill in the art to provide the desired formulation. Generally, the resulting solution will be apportioned into vials for lyophilization. Each vial contains a single dose (including, but not limited to, 10-1000 mg or 100-500 mg) or multiple doses of the ADC. The lyophilized powder may be stored under appropriate conditions, such as at about 4° C. to room temperature. In some embodiments, reconstitution of this lyophilized powder with water or other suitable carrier for injection provides a formulation for use in parenteral administration. For reconstitution, about 1-50 mg, about 5-35 mg, or about 9-30 mg of lyophilized powder is added per mL of sterile water or other suitable carrier. The exact amount can be adjusted and empirically determined.

[0044] As used herein, the term "lyophilized" refers to a composition that has been freeze-dried under vacuum. Lyophilization is typically accomplished by freezing a particular formulation so that the solute is separated from the solvent(s). The solvent is then usually removed by sublimation (i.e., primary drying) and then by desorption (i.e., secondary drying). In some embodiments, a lyophilization cycle consists of three steps: freezing, primary drying, and secondary drying. See, for example, AP Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977). In a typical freezing step, the solution is cooled to initiate the formation of ice. This step also induces the crystallization of the bulking agent. The ice sublimes in the primary drying stage, which is accomplished by lowering the chamber pressure below the vapor pressure of the ice, using a vacuum to promote sublimation, and introducing heat. Finally, the adsorbed or bound water is usually removed in the secondary drying stage under reduced chamber pressure and elevated shelf temperature. In some embodiments, the process produces a material known as a lyophilized cake. In various embodiments, the cake can be reconstituted with either sterile water or a suitable diluent for administration, as described herein.

[0045] In some embodiments, the pharmaceutical composition is stored in a glass vial, hi other embodiments, the pharmaceutical composition is stored in a polycarbonate bottle. anti-tissue factor antibody

[0046] The ADC in the pharmaceutical composition comprises an antibody that specifically binds to tissue factor (TF).

[0047] In certain embodiments, the antibody of the ADC specifically binds to the extracellular domain of human TF (SEQ ID NO: 39).

[0048] In certain embodiments, the antibody of the ADC comprises a heavy chain sequence. Illustrative heavy chain sequences are provided in Table 1. The heavy chain sequence may be from the antibody clone identified as 25A3.

[0049] In certain embodiments, the antibody of the ADC comprises a light chain sequence. Illustrative light chain sequences are provided in Table 1. The light chain sequence may be from the antibody clone identified as 25A3. [Table 1]

[0050] In certain embodiments, the antibody of the ADC comprises the V H In certain embodiments, the ADC administered in the methods of treating provided herein comprises the illustrative V H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H The antibody comprises the sequence.

[0051] In certain embodiments, the antibody of the ADC comprises the V L In certain embodiments, the antibodies of the ADCs administered in the methods of treatment provided herein comprise a VL sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the illustrative VL sequence of SEQ ID NO:38. L Contains arrays.

[0052] In certain embodiments, the antibody of the ADC comprises the V H Sequence, and V of SEQ ID NO:38 L Contains arrays.

[0053] In certain embodiments, the antibody of the ADC comprises the VH sequence of SEQ ID NO: 37 and the VL sequence of SEQ ID NO: 38. In certain embodiments, the antibody of the ADC comprises the illustrative VH sequence of SEQ ID NO: 37 and the VL sequence of SEQ ID NO: 38. H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H and VL sequences having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the illustrative VL sequence of SEQ ID NO:38. L and an array.

[0054] In certain embodiments, the antibody of the ADC comprises the heavy chain sequence of SEQ ID NO:40 and the light chain sequence of SEQ ID NO:41.

[0055] In some embodiments, the antibody of the ADC contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody retains the ability to bind to TF. In some embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). Antibodies generated by conservative amino acid substitutions are included in the present disclosure. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein determined. Conservative substitutions (e.g., within amino acid groups with similar properties and / or side chains) can be made to maintain properties or not significantly change properties. Optionally, the antibodies provided herein include post-translational modifications of the reference sequence.

[0056] In certain embodiments, the ADC comprises an anti-tissue factor antibody comprising heavy chain CDR sequences from antibody clone 25A3. The antibody 25A3 CDR sequences as determined by the Exemplary, Kabat, Chothia, AbM, Contact, and IMGT numbering systems are shown in Table 2. [Table 2]

[0057] In certain embodiments, the ADC comprises a CDR-H3 sequence that is 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the CDR-H3 sequence from antibody clone 25A3. In certain embodiments, the ADC comprises a CDR-H2 sequence that is 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the CDR-H2 sequence from antibody clone 25A3. In certain embodiments, the ADC comprises a CDR-H1 sequence that is 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the CDR-H1 sequence from antibody clone 25A3. In certain embodiments, the ADC comprises two heavy chain CDRs that are 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the corresponding two heavy chain CDRs from antibody clone 25A3. In certain embodiments, the ADC comprises three heavy chain CDRs that are 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the three heavy chain CDRs from antibody clone 25A3.

[0058] In certain embodiments, the ADC comprises an anti-tissue factor antibody comprising a light chain CDR from antibody clone 25A3. In certain embodiments, the ADC comprises a CDR-L3 sequence that is 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the CDR-L3 sequence from antibody clone 25A3. In certain embodiments, the ADC comprises a CDR-L2 sequence that is 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the CDR-L2 sequence from antibody clone 25A3. In certain embodiments, the ADC comprises a CDR-L1 sequence that is 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the CDR-L1 sequence from antibody clone 25A3. In certain embodiments, the ADC comprises two light chain CDRs that are 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the corresponding two light chain CDRs from antibody clone 25A3. In certain embodiments, the ADC comprises three light chain CDRs that are 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the three light chain CDRs from antibody clone 25A3.

[0059] In certain embodiments, the ADC comprises a CHR-H3 sequence that is 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the CDR-H3 sequence from antibody clone 25A3, and a CDR-L3 sequence that is 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the CDR-L3 sequence from antibody clone 25A3. In certain embodiments, the ADC comprises six CDR sequences that are 50%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the corresponding six CDRs from antibody clone 25A3.

[0060] Linker-toxin The ADCs provided herein comprise a cytotoxic agent, such as an auristatin derivative.

[0061] In certain embodiments, the auristatin derivative (toxin) is a moiety derived from compound 9 below. [ka]

[0062] In certain embodiments, the ADCs of the disclosure comprise a TF antibody conjugated to an auristatin derivative (toxin) via a linker (L). In certain embodiments, the ADC comprises (a) an antigen binding protein (Ab) that binds to the extracellular domain of human tissue factor (TF), wherein the Ab comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, where VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 are from the antibody designated 25A3, and (b) one or more linker-toxin moieties represented by formula (III): [ka] wherein ## represents the point of attachment of the linker-toxin moiety to the TF antibody, and the linker-toxin moiety is attached to the TF antibody via a covalent bond.

[0063] Antibody-drug conjugates In some embodiments, the ADCs provided herein have the following formula (I): [ka] or a pharma- ceutically acceptable salt thereof.

[0064] In certain embodiments, the Ab is a tissue factor (TF) antibody, and the Ab comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, and VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 are from the antibody designated 25A3.

[0065] In certain embodiments, n is an integer equal to or greater than 1. In certain embodiments, the succinimidyl group is attached to the Ab via a covalent bond. In further embodiments, the succinimidyl group is attached to the Ab via a cysteine ​​residue of the Ab.

[0066] In certain embodiments, n is selected from the group consisting of 1, 2, 3, and 4. In certain embodiments, n is selected from the group consisting of 3 and 4.

[0067] In certain embodiments, the Ab comprises a VH sequence that is SEQ ID NO:37 and a VL sequence that is SEQ ID NO:38.

[0068] In certain embodiments, Ab is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWVRQAPGQGLEWMGWIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDAGTYSPFGYGMDVWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP and a heavy chain sequence which is: CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 40); and a light chain sequence which is DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLLIYKAYNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLPPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41).

[0069] A collection of antibody-drug conjugates Also provided herein is a pharmaceutical composition comprising a population of antibody-drug conjugates (ADCs) provided herein, the ADCs having the following formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein the population is a mixed population of antibody-drug conjugates, where n varies from 1 to 4. In certain embodiments, the average n of the population is about 3.8.

[0070] In certain embodiments, the Ab is a tissue factor (TF) antibody, and the Ab comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, and VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 are from the antibody designated 25A3.

[0071] In certain embodiments, the Ab comprises a VH sequence that is SEQ ID NO:37 and a VL sequence that is SEQ ID NO:38.

[0072] In certain embodiments, Ab is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWVRQAPGQGLEWMGWIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDAGTYSPFGYGMDVWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP and a heavy chain sequence which is: CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 40); and a light chain sequence which is DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLLIYKAYNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLPPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41).

[0073] Drug-to-Antibody Ratio The number of linker toxin moieties conjugated to an antibody in an ADC is defined as the drug-antibody ratio or DAR. As known in the art, most conjugation methods produce ADC compositions containing various DAR species due to a mixed population of ADCs, and the reported DAR is the average of the individual DAR species (n is 1, 2, 3, 4, etc.). Therefore, when an ADC described herein is defined as having a specific DAR, it should be understood that the number provided represents the average of the individual DAR species in the ADC composition. In some embodiments, the DAR is measured by UV / vis spectroscopy, hydrophobic interaction chromatography (HIC), and / or reversed-phase liquid chromatography separation with time-of-flight detection and mass characterization (RP-UPLC / mass spectrometry). In some embodiments, the distribution of drug binding forms (e.g., proportions of DAR0, DAR1, DAR2, etc. species) can also be analyzed by various techniques known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC, or isoelectric focusing gel electrophoresis (IEF) (see, e.g., Sun et al., Bioconj Chem., 28:1371-81 (2017); Wakankar et al., mAbs, 3:161-172 (2011)).

[0074] The drug-antibody ratio (DAR) of the ADC in the compositions described herein may be about 1 to 4. In certain embodiments, the DAR is about 2 to 4. In certain embodiments, the DAR is about 3 to 4. In certain embodiments, the DAR is about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0. In certain embodiments, the DAR is about 3.8. therapeutic use

[0075] The pharmaceutical compositions provided herein can be administered to a human by infusion, for example, continuous infusion over a period of time.

[0076] The pharmaceutical compositions provided herein may be useful for treating a disease or disorder involving TF. In some embodiments, the disease or disorder is one that can benefit from treatment with an anti-TF antibody or ADC.

[0077] In some embodiments, the pharmaceutical compositions provided herein are provided for use as a medicament. In some embodiments, the pharmaceutical compositions provided herein are provided for use in the manufacture or preparation of a medicament. In some embodiments, the medicament is for the treatment of a disease or disorder that can benefit from an anti-TF antibody or ADC.

[0078] In some embodiments, provided herein are methods of treating a disease or disorder in a subject in need of such treatment by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0079] In some embodiments, the disease or disorder that may benefit from treatment with an anti-TF antibody or ADC is cancer. In some embodiments, the pharmaceutical compositions provided herein are provided for use as a medicament for the treatment of cancer. In some embodiments, the pharmaceutical compositions provided herein are provided for use in the manufacture or preparation of a medicament for the treatment of cancer. In some embodiments, a method of treating cancer is provided herein. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), urothelial carcinoma, ovarian cancer (e.g., epithelial), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., with squamous cell histology), and pancreatic cancer.

[0080] In some embodiments, provided herein is a method for delaying the onset of cancer in a patient who needs to delay the onset of cancer by administering to the subject an effective amount of the pharmaceutical composition provided herein.In some embodiments, provided herein is a method for performing late-stage interventional treatment of cancer in a subject who needs to do so.For example, the pharmaceutical composition can reduce tumor size in a subject who needs to reduce tumor size (e.g., tumor volume) or inhibit tumor growth in a subject who needs to inhibit tumor growth.

[0081] In some embodiments, provided herein are methods of preventing the onset of cancer in a patient in need thereof by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0082] In some embodiments, provided herein is a method of reducing the size of a tumor in a subject in need of such reduction (e.g., tumor volume) by administering to the subject an effective amount of a pharmaceutical composition provided herein. In some embodiments, the pharmaceutical composition provided herein reduces the tumor size (e.g., tumor volume) by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the ADCs provided herein inhibit tumor growth by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0083] In some embodiments, provided herein is a method of reducing the number of metastases in a subject in need thereof by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0084] In some embodiments, provided herein are methods of extending overall survival, median survival, or progression-free survival in a subject in need thereof by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0085] In some embodiments, provided herein are methods for treating a subject who has become resistant to a standard therapeutic agent by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0086] In some embodiments, the disease or disorder is a disease or disorder involving angiogenesis. In certain embodiments, the disease or disorder involving angiogenesis is cancer. In some embodiments, the disease or disorder is a disease or disorder involving vascular inflammation.

[0087] In some embodiments, the pharmaceutical compositions provided herein are provided for use as a medicament for the treatment of a disease or disorder involving angiogenesis. In some embodiments, the pharmaceutical compositions provided herein are provided for use in the manufacture or preparation of a medicament for the treatment of a disease or disorder involving neovascularization. In certain embodiments, the disease or disorder involving angiogenesis is cancer. In some embodiments, the pharmaceutical compositions provided herein are provided for use as a medicament for the treatment of a disease or disorder involving vascular inflammation. In some embodiments, the pharmaceutical compositions provided herein are provided for use in the manufacture or preparation of a medicament for the treatment of a disease or disorder involving vascular inflammation.

[0088] In some embodiments, provided herein is a method for treating a disease or disorder involving angiogenesis in a subject in need of such treatment by administering to the subject an effective amount of the pharmaceutical composition provided herein.In certain embodiments, the disease or disorder involving angiogenesis is cancer.In some embodiments, provided herein is a method for treating a disease or disorder involving vascular inflammation in a subject in need of such treatment by administering to the subject an effective amount of the pharmaceutical composition provided herein.

[0089] In some embodiments, provided herein is a method of delaying the onset of a disease or disorder involving angiogenesis in a subject in need thereof by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0090] In some embodiments, provided herein are methods of preventing the onset of a disease or disorder involving angiogenesis in a subject in need of such prevention by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0091] In some embodiments, provided herein is a method of delaying the onset of a disease or disorder involving vascular inflammation in a subject in need thereof by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0092] In some embodiments, provided herein are methods of preventing the onset of a disease or disorder associated with vascular inflammation in a subject in need of such prevention by administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0093] It is understood that modifications that do not substantially affect the activity of the various embodiments described herein are also provided within the definition of the subject matter described herein. Thus, the following examples are intended to illustrate, but not limit, the disclosure. EXAMPLES

[0094] The following examples are intended as illustrations of various embodiments disclosed herein. In certain embodiments, compounds are prepared by various synthetic routes. These examples are not intended, and should not be interpreted, as limiting the scope of the present invention. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0095] Example 1: Synthesis of Linker-Toxin A The following examples illustrate the preparation of exemplary linker-toxins (also referred to as linker-toxin A, LT-A), including compound 9, an auristatin derivative. [ka] 7.1 Ethyl (2R,3R)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanoate (Compound 1) [ka]

[0096] To a stirred solution of (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (Boc-Dap-OH, 4.31 g, 15.0 mmol) in absolute ethanol (27.0 mL) at 0° C., thionyl chloride (3.0 mL) was added dropwise. The resulting solution was allowed to warm to room temperature and progress was monitored by HPLC-MS. After 18 h, no remaining starting material was detected and the solution was concentrated to dryness under reduced pressure. The resulting oil was suspended in toluene (10 mL) and concentrated twice under reduced pressure, then suspended in diethyl ether (5 mL) and concentrated twice under reduced pressure to give the title compound as a white solid foam (3.78 g, % quantitative yield). MS m / z obs. = 216.5 (M+1). 7.2 (3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoic acid (compound 3) [ka]

[0097] Compound 2 was prepared as described in International Patent Application Publication No. WO2016 / 041082.

[0098] To a stirred solution of compound 2 (6.965 g, 14.14 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (5.0 mL). The reaction was monitored by HPLC-MS for completion and after 40 h no starting material remained. The reaction was concentrated under reduced pressure and co-evaporated with toluene (2×10 mL) and dichloromethane (2×10 mL) to give a foamy white solid (6.2 g, quantitative yield with residual TFA). This material was dissolved in 200 mL of hot EtOAc:hexanes (1:3) and allowed to cool to room temperature. During cooling a precipitate formed as well as some small crystals. 5 mL of EtOAc was added and the suspension was heated once more to completely dissolve the precipitate. Upon cooling to room temperature more crystals formed and the flask was left at −30° C. overnight. The next morning the mother liquor was decanted and the crystals were rinsed with 2×50 mL of hexanes and dried under high vacuum. 5.67 g of the title compound was recovered as a crystalline product. MS m / z obs.=405.7(M+1). 7.3 Ethyl (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate (compound 4) [ka]

[0099] To a stirred solution of compound 3 (6.711 g, 15.37 mmol, 1.025 equiv.) in a mixture of dichloromethane (5.0 mL) and N,N-dimethylformamide (5.0 mL) was added HATU (5.732 g, 15.07 mmol, 1.005 equiv.) and N,N-diisopropylethylamine (7.84 mL, 3 equiv.) at room temperature. After stirring for 30 min at room temperature, a solution of compound 1 (3.776 g, 15.00 mmol, 1.0 equiv.) in a mixture of dichloromethane (1.0 mL) and N,N-dimethylformamide (1.0 mL) was added dropwise and rinsed in residual compound 1 with an additional 3 mL of dichloromethane:N,N-dimethylformamide (1:1). The reaction was monitored by HPLC-MS and no remaining compound 1 was observed after 15 min. The reaction was concentrated under reduced pressure, diluted with ethyl acetate (ca. 125 mL) and the organic phase was extracted with 1M HCl (2 x 50 mL), 1 x dH2O (1 x 50 mL), saturated NaHCO3 (3 x 50 mL), and brine (25 mL). Both the acidic and basic aqueous layers were washed with 25 mL of EtOAc. All organics were then pooled, dried over MgSO4, filtered, and concentrated to give a red oil. The residue was dissolved in a minimum amount of dichloromethane (ca. 10 mL) and loaded onto a Biotage® SNAP Ultra 360 g silica gel column (Isolera™ Flash System, Biotage AB, Sweden) for purification (20-100% EtOAc in hexanes over 10 column volumes). Fractions containing pure product were pooled to recover 7.9 g of a foamy white solid. The impure fractions were subjected to a second purification on a Biotage® SNAP Ultra 100 g silica gel column and pooled with the pure product to recover the title compound as a white foam solid (8.390 g, 88.3%). MS m / z obs.=634.7 (M+1). 7.4 (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (compound 5) [ka]

[0100] To a stirred solution of compound 4 (8.390 g, 13.24 mmol) in 1,4-dioxane (158 mL) was added dH2O (39.7 ml) and lithium hydroxide monohydrate (1 M in H2O, 39.7 mL, 3 equiv.). The reaction was stirred at 4° C. and monitored by HPLC-MS for consumption of starting material, which took 3 days before only traces of compound 4 remained. During the course of the reaction, in addition to the desired material, a small proportion of a new product was formed, corresponding to loss of methanol (β-elimination, <2%). The reaction was acidified by addition of 1 M aqueous HCl (50 mL) and concentrated under reduced pressure to remove dioxane. The remaining reaction mixture was extracted with ethyl acetate (4×50 mL) and the organic phases were pooled, washed with brine (15 mL+2 mL of 2 M HCl), dried over MgSO4, filtered, and concentrated under reduced pressure to give a pale oil. The oil was redissolved in diethyl ether (ca. 50 mL) and concentrated under reduced pressure (3x) to aid in the removal of residual dioxane to give the title product as a hard oil (7.81 g, 97% yield with some residual dioxane and compound 4). MS m / z obs.=606.7 (M+1). 7.5 Benzyl ((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((4-(2,2,2-trifluoroacetamido)phenyl)sulfonamido)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (compound 7) [ka]

[0101] Compound 6 was prepared as described in International Patent Application Publication No. WO2016 / 041082.

[0102] To a stirred solution of compound 5 (7.12 g, 11.754 mmol) in dichloromethane (20 mL), 2,2,2-trifluoro-N-(4-sulfamoylphenyl)acetamide (compound 6, 4.095 g, 1.3 eq., dissolved in 3 mL of DMF), N,N-dimethylpyridine (1.867 g, 1.3 eq.), and N,N-dimethylformamide (1.5 mL) were added to produce a pale yellow suspension. An additional addition of 5 mL of DMF did not clear the solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) (2.817 g, 1.25 eq.) was added in one portion and the reaction was monitored by HPLC-MS. After 48 h, the reaction was no longer proceeding and an additional 400 mg of EDCI was added. After 18 hours, no remaining starting material was observed and the reaction was concentrated under reduced pressure to give a yellow oil. The oil was dissolved in ethyl acetate (ca. 150 mL) and 1M HCl (20 mL) and the organic phase was washed with cold 2M HCl (2×10 mL), saturated NaHCO3 (1×10 mL), and brine (20 mL + 5 mL of 2M HCl). The acidic and basic aqueous fractions were extracted with EtOAc (1×20 mL) and all organic fractions were pooled, dried over MgSO4, and concentrated under reduced pressure to give a crude oily solid (13 g). The residue was dissolved in dichloromethane (ca. 10 mL) and loaded onto a Biotage® SNAP Ultra 360 g silica gel column and purified under a 10-100% EtOAc (2% AcOH) gradient in hexanes over 12 column volumes, with a 3 column volume plateau at 50% EtOAc. Fractions containing pure product were pooled, concentrated under reduced pressure, dissolved and concentrated from toluene (2×10 mL) and diethyl ether (2×10 mL) to give 7.1 g of the desired product as a white foam solid. Impure fractions were subjected to repeated purification under less steep gradient conditions using a Biotage® SNAP Ultra 100 g silica gel column on an Isolera™ apparatus. All pure fractions were pooled to recover the pure product (title compound) as a white foam solid (8.60 g, 86%). MS m / z obs.=856.7 (M+1).

[0103] 7.6(S)-2-Amino-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((4-(2,2,2-trifluoroacetamido)phenyl)sulfonamido)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide (compound 7a) [ka] Compound 7 (3.71 g, 4.33 mmol) was dissolved in 10% N,N-dimethylformamide in ethyl acetate (30 mL) in a round bottom flask containing a magnetic stirrer and fitted with a 3-way gas line adapter. The vessel was evacuated under reduced pressure twice and backfilled with nitrogen gas. 10% palladium on carbon (0.461 g, 0.1 equiv.) was added in one portion, a 3-way adapter was fitted to the flask, a hydrogen balloon was attached to the adapter, and the vessel was evacuated under reduced pressure twice and backfilled with hydrogen. The reaction was stirred for 2 days, during which the hydrogen balloon was occasionally refilled. After approximately 48 hours, HPLC-MS analysis indicated no starting material remained. The reaction was diluted with methanol (20 mL) and filtered through a plug of Celite. The Celite was washed with methanol (2×50 mL). All filtrates were pooled and concentrated under reduced pressure to dissolve the resulting oil, which was concentrated from dichloromethane. After drying under reduced pressure, the title compound was isolated as a colorless powder (3.10 g, 99%). MS m / z obs.=722.6 (M+1). 7.7 (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((4-(2,2,2-trifluoroacetamido)phenyl)sulfonamido)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide (compound 8) [ka]

[0104] To a stirred solution of N,N-(L)-dimethylvaline (1.696 g, 9.35 mmol) in N,N-dimethylformamide (10 mL) was added HATU (3.216 g, 8.46 mmol) and di-isopropylethylamine (3.10 mL, 17.8 mmol). After 5 min a clear yellow solution was obtained. Stirring was continued for an additional 10 min, then compound 7a (3.213 g, 4.45 mmol) was added in one portion. After stirring for an additional 1 h, HPLC-MS showed traces of compound 7a remained, and the reaction was 16 h. The reaction was then concentrated under reduced pressure, diluted with ethyl acetate (120 mL) and 40 mL of NaHCO3 (sat.):5% LiCl (1:1), and transferred to a separatory funnel. The aqueous layer was removed and the organic phase was washed with LiCl (1×20 mL), NaHCO3 (sat., 2×20 mL). The aqueous layers were pooled and extracted with EtOAc (3×50 mL). The organic layers were pooled, washed with brine (1×20 mL), dried over sodium sulfate, filtered, concentrated to give a DMF-containing oil and concentrated via rotary evaporator to remove residual DMF to give 7 g of a crude pale yellow oil. The oil was dissolved in 10% methanol in a minimal amount of dichloromethane (approximately 11 mL) and loaded onto a Biotage® SNAP Ultra 360 g silica gel column for purification (2-20% MeOH in CH2Cl2 over 15 column volumes, product elutes at approximately 10-13%). Fractions containing the desired product were pooled and concentrated under reduced pressure to give the title compound as a colorless foam. The impure fractions were combined, evaporated and subjected to repeated purification on a Biotage® SNAP Ultra 100 g silica gel column on an Isolera™ apparatus and combined with the pure product from the first column to give a colorless foamy solid (3.78 g). MS m / z obs.=850.6 (M+1). 7.8 (S)-N-((3R,4S,5R)-1-((S)-2-((1R,2R)-3-((4-aminophenyl)sulfonamido)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide (compound 9) [ka]

[0105] To a stirred solution of compound 8 (0.980 g, 1.154 mmol) in 1,4-dioxane (15 mL) was added water (3.5 mL) and 1 M lithium hydroxide monohydrate (3 eq, 3.46 mL). The resulting pale suspension was stirred at 4° C. and monitored by HPLC-MS for consumption of starting material. When conversion was complete (approximately 5 days), the reaction was neutralized with 3.46 mL of 1 M HCl and concentrated under reduced pressure to remove dioxane. The resulting aqueous phase was diluted with 60 mL of EtOAc and 5 mL of brine, then extracted with ethyl acetate (2×30 mL). The organic fractions were pooled, dried over Na2SO4, filtered, and evaporated to give the title compound as a tan solid (0.930 g). R f = 0.5 (8% MeOH in CH2Cl2). MS m / z obs. = 753.7 (M+1). 7.9 2,3,5,6-Tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate (Compound 15) [ka]

[0106] In a dry 50 mL conical flask, 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid (compound 14, 1.000 g, 4.52 mmol) and maleic anhydride (0.443 g, 4.52 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL). The reaction was stirred at room temperature under N2 for 6 h, then cooled to 0 °C and syn-collidine (1.263 mL, 2.1 equiv.) was added dropwise. In a separate dry 50 mL conical flask, tetrafluorophenol (3.002 g, 4 equiv.) was dissolved in anhydrous N,N-dimethylformamide (10 mL). The flask was cooled to 0 °C in an ice bath and trifluoroacetic anhydride (2.548 mL, 4 equiv.) was added dropwise. After stirring for 15 min, syn-collidine (2.407 mL, 4 eq) was added dropwise. The flask was stirred for an additional 15 min, then the contents were added dropwise via syringe to the first flask. The reaction was allowed to warm to room temperature and stirring was continued under N2. The reaction was monitored by HPLC-MS for consumption of starting material. After 6 days, the reaction was complete with total consumption of compound 14, leaving only compound 15 and a small amount (~5%) of the bis-TFP maleamide intermediate. The reaction was transferred to a separatory funnel, diluted with diethyl ether (75 mL), and washed with 5% LiCl (1 x 20 mL), 1 M HCl (2 x 20 mL), saturated NaHCO3 (5 x 20 mL), and brine (1 x 20 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give a brown crude oil with residual DMF. The crude oil was dissolved in 8 mL of DMF:H2O+0.1% TFA (1:1), loaded onto a 60 g Biotage® SNAP Ultra C18 column (Biotage AB, Uppsala, Sweden) and purified under a linear 30-100% gradient of ACN / H2O+0.1% TFA over 8 column volumes. Pure fractions were pooled, diluted with brine (20 mL) and then extracted with 3×50 mL of Et2O. The pooled organics were dried over MgSO4, filtered and evaporated to recover the title compound as a pale yellow oil (1.34 g, 66% yield). 7.10 tert-Butyl ((S)-1-(((S)-1-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (compound 12) [ka]

[0107] Compound 11 was prepared as described in International Patent Application Publication No. WO2016 / 041082.

[0108] To an empty 25 mL pear-shaped flask was added compound 11 (1.342 g, 3.58 mmol, 3.0 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.664 g, 3.46 mmol, 2.9 equiv.), and 7-hydroxy-azabenzotriazole (HOAT) (0.472 g, 3.46 mmol, 2.9 equiv.). These solids were dissolved in a mixture of N,N-dimethylformamide (0.5 mL) and dichloromethane (4.5 mL) with stirring at room temperature for 30 minutes. Separately, compound 9 (0.900 g, 1.20 mmol) was dissolved in a mixture of N,N-dimethylformamide (0.2 mL) and dichloromethane (1.8 mL) and added to the pear-shaped flask and rinsed with dichloromethane (1.0 mL). The stirring speed was increased to 1000 rpm to create a vortex. Within 2 min of adding compound 9, copper(II) chloride (0.514 g, 3.83 mmol, 3.2 equiv) was added neat in one portion via a narrow powder funnel into the center of the vortex. The initially light yellow solution turned into a dark brown suspension, which changed to a dark green suspension over 10 min. The reaction was monitored for completion by HPLC-MS, and no change in reaction progress was observed between samples taken at 30 min and 1 h (approximately 95% complete). The reaction was stirred overnight at room temperature, then 2-(2-aminoethylamino)ethanol (0.483 mL, 4.781 mmol, 4 equiv), EtOAc (10 mL), and dH2O (5 mL) were added to the stirred suspension, causing a color change to dark blue. The suspension was stirred vigorously for 4 h, during which the suspended solids gradually dissolved into a biphasic mixture. The mixture was transferred to a separatory funnel, diluted with EtOAc (100 mL) and brine (10 mL), and the aqueous layer was extracted using 10% IpOH / EtOAc (4×50 mL). The organic layers were pooled, washed with brine (10 mL), dried over Na2SO4, and evaporated to give a crude faint blue solid. The crude solid was dissolved in a mixture of methanol (0.5 mL) and dichloromethane (6 mL) and purified on a Biotage® SNAP Ultra 100 g silica gel column (2-20% MeOH in CH2Cl2 over 10 column volumes, followed by an 8 column volume plateau at 20% MeOH).After 1-2 column volumes with approximately 20% MeOH in CH2Cl2, the product eluted as a broad peak. Fractions containing the desired material were pooled and concentrated under reduced pressure to give the title compound as a white solid (1.105 g, 83%). MS m / z obs.=555.9((M+2) / 2), 1109.8(M+1). 7.11 (S)-2-((S)-2-amino-3-methylbutanamide)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-5-ureidopentanamide (compound 13) [ka]

[0109] To a solution of compound 12 (0.926 g, 0.834 mmol) was added a mixture of dichloromethane (10 mL) and trifluoroacetic acid (2.0 mL). The reaction was monitored by HPLC-MS for consumption of starting material (approximately 45 min). The reaction was coevaporated under reduced pressure with acetonitrile (2×10 mL) and dichloromethane (2×10 mL) to remove excess trifluoroacetic acid. The resulting residue was dissolved in a minimum amount of dichloromethane and methanol (3:1, v / v, approximately 2 mL) and added dropwise via pipette to a stirred solution of diethyl ether (200 mL) and hexanes (100 mL) to produce a suspension of pale white solid. The solid was filtered and dried under vacuum to give the title compound in the form of a white powder as the trifluoroacetate salt (1.04 g, quantitative yield with some residual solvent). MS m / z obs.=505.8((M+2) / 2). 7.12 (S)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-2-((S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-14-isopropyl-12-oxo-3,6,9-trioxa-13-azapentadecanamido)-5-ureidopentanamide (Linker-Toxin A) [ka]

[0110] To a stirred solution of compound 13 (0.722 g, 0.584 mmol) in N,N-dimethylformamide (4 mL) was added compound 15 (0.314 g, 1.2 equiv.) and diisopropylethylamine (0.305 mL, 3.0 equiv.). HPLC-MS analysis at 2 h showed no remaining starting material. The reaction was acidified with TFA (300 μL) and then diluted with diH2O+0.1% TFA (9 mL). The resulting solution was loaded onto a 120 g Biotage® SNAP Ultra C18 column (Biotage, Uppsala, Sweden) and purified under an ACN / H2O+0.1% TFA gradient: 20-60% ACN over 10 column volumes, 60-100% ACN over 5 column volumes. The product eluted around 40% ACN. Pure fractions identified by LCMS were pooled and lyophilized. A white powder solid was recovered from the freeze dryer. The freeze drying was repeated at a higher concentration in a vial (approximately 50 mg / mL in 2:1 HO / ACN) to produce the title compound as a denser, less aggregated freeze dried solid (754.2 mg, 91%). MS m / z obs.=647.4((M+2) / 2), 1292.8(M+1). Example 2: ADC synthesis

[0111] An antibody-drug conjugate (ADC) of anti-TF antibody and the linker-toxin A prepared in Example 1 was prepared as described below.

[0112] Briefly, 5-10 mg / mL of 25A3 antibody (see Tables 1 and 2 for CDR and V-region sequences of clone 25A3) in phosphate buffered saline (PBS), pH 7.4, was reduced by the addition of tris(2-carboxyethyl)phosphine (2.0-2.5 or 3.2 molar equivalents) and diethylenetriaminepentaacetic acid to a final concentration of 0.8 mM. After 2 hours at 37°C, the partially reduced antibody was chilled on ice for 10 minutes and then conjugated with 8 molar equivalents of linker-toxin A on ice for 1 hour. The reaction was quenched with excess N-acetyl-L-cysteine. The quenched reaction was placed on ice for 30 minutes before purification. The ADC was purified through two rounds of 40 kDa MWCO Zeba™ spin desalting columns (10 mL columns, product number 8772, lot number RL240689), each according to the manufacturer's protocol. Prior to purification, both column sets were primed with sterile PBS. The ADC was first purified through one set of PBS primed columns, then a sample was collected and purified a second time through the other set. After the second purification, the ADC were pooled back together, sterile filtered and frozen at -80°C.

[0113] The drug-antibody ratio (DAR) may be measured by UV / vis spectroscopy, hydrophobic interaction chromatography (HIC), and / or reversed-phase liquid chromatography separation with time-of-flight detection and mass characterization (RP-UPLC / mass spectrometry), as described in WO2016 / 041082. The distribution of drug conjugated forms (e.g., proportions of DAR0, DAR1, DAR2, etc. species) may also be analyzed by a variety of techniques known in the art, including MS (with or without an associated chromatographic separation step), hydrophobic interaction chromatography, reversed-phase HPLC, or isoelectric focusing gel electrophoresis (IEF), as also described in WO2016 / 041082.

[0114] In this example, the drug-antibody ratio (DAR) of the resulting ADC was approximately 3. DAR was determined by hydrophobic interaction chromatography. Average DAR=(0×(DAR0 area%)+2×(DAR2 area%)+4×(DAR4 area%)+6×(DAR6 area%)+8×(DAR8 area%) / 100. Size exclusion chromatography was used to ensure that the ADC preparation was at least 95% monomeric.

[0115] For example, the ADC comprising 25A3 and LT-A (25A3-LT-A) prepared in this example was used in the studies in Example 3 below. Example 3: Formulation Development

[0116] The purpose of this formulation development study was to investigate the effect of buffer type, concentration, and pH on the stability of the solution formulation. The goal was to develop a stable frozen solution of 25A3-LT-A as prepared in Example 2.

[0117] Preparation of solution formulations

[0118] A total of 15 different solution formulations were tested in the study. The buffer preparations are shown in Table 3, and three different buffers-histidine, phosphate, and citrate were prepared at three concentrations-10 mM, 30 mM, and 50 mM, and at three pH levels: pH 5, pH 6, and pH 7. Formulations 1-15 in Table 3 were prepared from a 4.6 mg / mL aqueous buffer solution of 25A3-LT-A. The 25A3-LT-A solution was concentrated and dialyzed against the designed buffer. The final formulations were adjusted to concentrations close to 20 mg / mL when possible. The 25A3-LT-A formulations were filtered using a 0.2 μm syringe disc filter. [Table 3]

[0119] Formulation stability analysis

[0120] The stability of each formulation was tested in individual vials containing 1 mL of solution. The vials were left at temperatures ranging from -80°C to 40°C for up to 6 months. UV / Vis, size exclusion chromatography (SEC), dynamic light scattering (DLS), and imaging capillary electrophoresis (iCE) analyses were performed at T0 and at each time point listed in Table 4. [Table 4]

[0121] Test 1: Concentration measurement by UV / Vis

[0122] Protein concentration was measured by UV / Vis at 280 nm for drug product formulations using a 125 μL control sample (L / N E01-10). The concentration of each formulation was tested at T0 and after storage at the conditions shown in Table 4. The results in Table 5 showed that the concentration of the formulations remained near the expected value of 20 mg / mL for each formulation throughout the study. [Table 5]

[0123] Test 2: Size heterogeneity analysis by SEC

[0124] The molecular size distribution of 25A3-LT-A was evaluated by SEC. The mobile phase was prepared using sodium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate and adjusted to pH 6.8. A blank was prepared by mixing 77 μL of 30 mM histidine buffer prepared at pH 6.0 with 123 μL of mobile phase to match the dilution used for the reference sample. The sample was mixed with the mobile phase and processed in the same manner as the blank.

[0125] The formulations showed different degrees of decrease in percent monomer and increase in percent high molecular weight (HMW) species under different storage conditions. For example, formulation 9, prepared in 50 mM phosphate buffer at pH 7, showed a 0.6% decrease in monomer and a 0.7% increase in HMW after 6 months of storage at -20°C. In contrast, this formulation showed a 2.4% decrease in monomer and a 2.5% increase in HMW after 6 months of storage at -80°C.

[0126] Formulations prepared with histidine stored at 2°C-8°C for up to 2 months showed no significant changes in percent monomer, percent HMW, or percent low molecular weight (LMW) species during storage. No significant changes were observed upon storage at 25°C / 60% RH for up to 2 weeks. However, changes in the SEC data were evident for formulations stored at 40°C / 75% RH for up to 2 weeks, with the exception of the histidine formulation. Figures 1A-1C show that the formulations prepared with histidine (Figure 1A) had less change in species percentage than the formulations prepared with citric acid (Figure 1B) and phosphoric acid (Figure 1C).

[0127] The significance of the difference in percent monomer between TO and after 2 weeks storage at 40°C was further examined statistically by one-way ANOVA. The analysis was performed based on buffer type, buffer concentration, and formulation pH, respectively. The results show that (1) formulations prepared with citrate and histidine had less change in percent monomer than formulations prepared with phosphate (Figure 2A), (2) the 30 mM concentration showed less change in percent monomer than the 10 mM and 50 mM concentrations (Figure 2B), and (3) smaller differences in percent monomer occurred at pH 5 and pH 6 (Figure 2C).

[0128] The data suggested that the ADCs provided herein were more unstable in phosphate buffer, therefore, statistical analysis was reexamined only for formulations prepared with citric acid and histidine. Figure 2D showed that formulations prepared with histidine showed less change in percent monomer upon storage at 40°C for up to 2 weeks. Statistics showed that samples stored at 40°C showed no significant difference in percent monomer based on buffer concentration or pH.

[0129] In summary, based on the SEC data of percent monomer change in formulations stored at 40°C / 75% RH for up to 2 weeks, higher molecular stability was observed when formulations were prepared at pH below 7 using histidine buffer and had buffer concentrations between 10 mM and 50 mM. Formulations 3, 9, and 12 also showed changes in percent monomer during storage at -20°C and -80°C for up to 6 months. The observations were consistent as formulations 3 and 9 were prepared with the less stable phosphate buffer. The change in formulation 12, prepared at pH 5 with 50 mM histidine buffer, may suggest further adjustment in the histidine concentration.

[0130] Test 3: Charge non-uniformity analyzed by iCE

[0131] Charge heterogeneity of 25A3-LT-A was evaluated using iCE. iCE dilutions were prepared by mixing Pharmalyte 3-10, Pharmalyte 8-10, Pharmalyte 5-8, 1% methylcellulose, pI marker 5.85, pI marker 9.50, and 10M urea in a Falcon tube. Samples were prepared in duplicate by adding 10 μL of sample to 90 μL of dilution. All iCE comparisons were shown in line graphs. The main pI peak shift percentage indicated the charge heterogeneity change of 25A3-LT-A.

[0132] Among the formulations prepared with histidine, no significant changes in percent main peak, percent acidic species, or percent basic species were observed during storage at -20°C and -80°C for up to 6 months. However, formulation 1 prepared at pH 7 with 50 mM histidine showed a consistent decrease in percent main peak (Figure 3). Formulation 1 also showed a decrease in percent main peak and percent acidic species during storage at 2°C to 8°C for up to 2 months. Thus, the results suggested that formulations with 50 mM histidine at pH 7 may be less stable. This was consistent with the SEC analysis, which also suggested less stability in formulation 12 prepared at pH 5 with 50 mM histidine buffer.

[0133] Formulations prepared with citric acid or phosphoric acid showed changes in the percent main peak, percent acidic species, or percent basic species during storage at 2°C-8°C for up to 2 months. As shown in Figure 4, the percent acidic species increased for all formulations prepared with citric acid during storage at 2°C-8°C for up to 2 months. Citric acid formulations 4 and 10 showed an approximately 2% decrease in the percent main peak. An increase in acidic species was also observed for all formulations prepared with phosphate buffer. Figure 5 showed that all formulations prepared with phosphoric acid, especially formulations 3 and 9, showed a decrease in the percent main peak during storage at 2°C-8°C for up to 2 months. Formulations 3 and 9 were prepared at pH 7 using buffer concentrations of 10 mM and 50 mM, respectively. These results again suggested that pH 7 may not be a condition that promotes stability of the formulations.

[0134] One-way ANOVA was applied to examine the changes in main peak percentage and acidic species percentage for formulations stored at 2°C-8°C for up to 2 months. The analysis suggested that formulations prepared with citrate buffer showed less change in main peak percentage and acidic species percentage. The data also suggested that less change in main peak percentage was observed for formulations stored at 2°C-8°C for up to 2 months when the buffer concentration was 30 mM (Figure 6). In addition, formulations prepared at pH 5-pH 6 at the same storage conditions showed less change in main peak percentage (Figure 7).

[0135] Under conditions of storage at 25°C for up to 2 weeks, all formulations prepared with citrate or phosphate buffers showed an increase in the percent acidic species. Formulations 1 and 11 prepared at pH 7 showed a significant decrease in the percent main peak. In a one-way ANOVA, the data suggested that no differences in the percent main peak were observed between phosphate and citrate buffers based on buffer type or buffer concentration. The data indicate that the expected changes were less in formulations prepared with 30 mM citrate buffers with a pH of 5-6. Also, among the formulations prepared with citrate or phosphate buffers, the analysis showed that the formulation prepared at pH 5 showed less change (Figure 8). These results suggested that a buffer concentration of 30 mM at pH 5-6 may be a better condition to stabilize the formulation.

[0136] During storage at 40°C for up to 2 weeks, all formulations prepared with phosphate buffer showed a significant decrease in the main peak percent. All formulations showed an increase in iCE acidic peak percent and a decrease in iCE basic peak percent. In contrast, formulation 12 prepared with histidine and formulation 6 prepared with citric acid showed less change in the main peak percent during storage at 40°C for up to 2 weeks. Both formulations were prepared at pH 5 using 50 mM buffer concentration. The results suggested that 50 mM histidine or citrate buffer at pH 5 may be better conditions to stabilize the formulations.

[0137] One-way ANOVA was applied to examine the difference in the main peak percentage between TO and after up to 2 weeks storage at 40°C. The data suggested that less change in the iCE main peak percentage was observed for formulations prepared in citrate or histidine buffer at pH 5 and 6. No significant differences in the analysis were observed based on buffer concentration. Also, formulations prepared in citrate or histidine buffer at pH 5 or 6 showed the least change after up to 2 weeks storage at 40°C.

[0138] In summary, the iCE charge heterogeneity data suggested that formulations prepared in histidine buffer at pH 5 or 6 showed minimal variation. The buffer concentration did not significantly affect the analysis, but the data suggested that a buffer concentration of 30 mM may be an appropriate concentration as a midpoint for the formulation.

[0139] Test 4: Particle size analyzed by DLS

[0140] DLS was used to assess the changes in mean particle size and percent polydispersity of each formulation at T0 and after storage. Each sample vial was gently agitated by vortexing for approximately 10 seconds and loaded onto the sample plate. Samples were analyzed using 10 acquisitions with triplicate measurements for each analysis. Changes were considered significant if the mean particle size and / or polydispersity were two- or three-fold over the storage period. Particle size distributions can also be considered significant if they became multimodal.

[0141] No significant changes in mean particle size or percent polydispersity were observed for the formulations tested, except for formulations 1 and 4 (Figure 9). The particle size distribution of formulation 4 became multimodal after 6 months of storage at -80°C and -20°C. Formulation 1 became multimodal after 4 and 6 months of storage at -20°C. Formulation 4 was prepared with 10 mM citric acid at pH 6. Formulation 1 was prepared with 50 mM histidine at pH 7. The results were consistent with the observation that formulation 1 was less stable in the iCE analysis. A significant increase in percent polydispersity in formulation 8 was observed after up to 2 weeks of storage at 40°C. Formulation 8 was prepared with 10 mM phosphate at pH 6. No significant changes in mean particle size or percent polydispersity were observed for the other formulations after up to 2 weeks of storage at 40°C.

[0142] In conclusion, the data suggested that histidine buffers provided the best stability for the molecule within a pH range of 5 to 6. Buffer concentrations appeared to be acceptable within the range of 10 mM to 50 mM.

[0143] From the above formulation studies based on the preparation and analysis of numerous formulations with variations in pH (5, 6, and 7), excipients (phosphate, citrate, and histidine), and concentrations (10 mM, 30 mM, 50 mM), the final formulation (Table 6) was selected with 20 mM histidine, pH 5.5 along with the addition of sucrose and polysorbate 80, and demonstrated surprisingly good stability. Histidine acts as a buffering agent and hydrochloric acid is used for pH control. Sucrose acts as an isotonicity agent and stabilizes the ADC in solution during storage. Polysorbate 80 is a surfactant and also stabilizes the injection during manufacturing and storage. [Table 6] Example 4: Stability of 25A3-LT-A Dosage Forms

[0144] 25A4-LT-A compositions were prepared as described in Table 6. The stability of the prepared compositions was tested under various storage conditions such as (i) <-65°C (first long-term storage condition), (ii) -20°C ± 5°C (second long-term storage condition), (iii) 25 ± 2°C / 60 ± 5% relative humidity (RH) (first accelerated storage condition), (iv) 5 ± 3°C (second accelerated storage condition), (v) about 25°C / 60% RH (third accelerated storage condition), and (vi) 40°C / 75% RH (harsh storage condition). Various storage periods such as 0 days (initiation), 1 day, 1 week, 2 weeks, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and 23 months have been investigated, while additional storage periods of 24 months, 30 months, 36 months, 48 ​​months, and 60 months are under investigation.

[0145] No significant changes in pH, protein concentration (tested by UV), antigen binding (tested by enzyme-linked immunosorbent assay, ELISA), purity (tested by high performance liquid chromatography, HPLC, or capillary electrophoresis sodium dodecyl sulfate, CE-SDS), charge variants (tested by imaging capillary isoelectric focusing, icIEF), aggregation or size variants (tested by size exclusion chromatography, SEC), or other attributes of the composition were observed for up to 23 months at storage conditions of <-65°C. The data also showed no change over 23 months at the proposed long-term storage temperature of -20°C. In addition, data generated at the intended storage conditions did not show significant trends over time for any of the tested attributes. For example, the DAR of the composition remained at approximately 3.8.

[0146] Several of the embodiments have been described herein. However, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A pharmaceutical composition comprising: (i) An antibody-drug conjugate of formula (I): 【Chemistry 19】 or a pharma- ceutically acceptable salt thereof, During the ceremony, the Ab is a tissue factor (TF) antibody, said Ab having the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the antibody designated 25A3; the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3; n is an integer of 1 or more, the antibody-drug conjugate, or a pharma- ceutically acceptable salt thereof, wherein a succinimidyl group is attached to the Ab via a covalent bond; (ii) a buffer solution; and (iii) a tonicifier; and (iv) a surfactant.

2. The pharmaceutical composition of claim 1 , wherein the succinimidyl group is attached to the Ab via a cysteine ​​residue of the Ab.

3. 3. The pharmaceutical composition of claim 1, wherein n is selected from the group consisting of 1, 2, 3, and 4, and optionally n is selected from the group consisting of 3 and 4.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the antibody-drug conjugate has a drug-to-antibody ratio (DAR) of about 1 to about 4, optionally about 2 to about 4, further optionally about 3, and even further optionally about 3.

8.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the Ab comprises a VH comprising the amino acid sequence of SEQ ID NO:37 and a VL comprising the amino acid sequence of SEQ ID NO:

38.

6. The Ab is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWVRQAPGQGLEWMGWIAPYSGNTN YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDDTAVYYCARDAGTYSPFGYGMDVWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA a heavy chain comprising the amino acid sequence of: PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 40); and a light chain comprising the amino acid sequence of: DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLLIYKAYNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLPPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:41).

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the buffer is histidine, the tonicity agent is sucrose, the surfactant is polysorbate 80, and the pharmaceutical composition further comprises hydrochloric acid.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, comprising 5-20 mg / mL of the antibody-drug conjugate, 10-50 mM histidine, 5-10% (w / v) sucrose, and 0.01-0.05% (w / v) polysorbate 80, wherein the pharmaceutical composition has a pH value of 5 to 6.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, comprising about 10 mg / mL of the antibody-drug conjugate, about 20 mM histidine, about 8% (w / v) sucrose, and about 0.02% (w / v) polysorbate 80, wherein the pharmaceutical composition has a pH value of about 5.

5.

10. A pharmaceutical composition according to any one of claims 1 to 9 for use in treating a disease or disorder in a subject.

11. A method of treating a disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition according to any one of claims 1 to 9.

12. 12. The pharmaceutical composition of claim 10 or the method of claim 11, wherein the disease or disorder is cancer, optionally selected from the group consisting of non-small cell lung cancer (NSCLC), urothelial carcinoma, ovarian cancer (e.g., epithelial), cervical cancer (e.g., having squamous cell or adenocarcinoma histology), head and neck cancer (e.g., having squamous cell histology), and pancreatic cancer.

13. The pharmaceutical composition or method according to any one of claims 10 to 12, wherein the subject is a human subject.

14. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition is lyophilized.

15. The pharmaceutical composition according to any one of claims 1 to 9 and 14, wherein the pharmaceutical composition is stored in a glass vial or a polycarbonate bottle.

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