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WO2025145034A1 - Anti-psma conjugates and methods of using the same - Google Patents

Anti-psma conjugates and methods of using the same Download PDF

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Publication number
WO2025145034A1
WO2025145034A1 PCT/US2024/062094 US2024062094W WO2025145034A1 WO 2025145034 A1 WO2025145034 A1 WO 2025145034A1 US 2024062094 W US2024062094 W US 2024062094W WO 2025145034 A1 WO2025145034 A1 WO 2025145034A1
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Prior art keywords
psma
conjugate
tumor
antibody
ald101
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French (fr)
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Esteban Cvitkovic
Morris Rosenberg
Tae Han
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TOAD Oncology Sa
Genmab AS
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TOAD Oncology Sa
Genmab AS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Prostate cancer ranks among the top for the number of new cancer cases in men in the United States.
  • surgery and radiotherapy are usually the options.
  • surgical or chemical androgen deprivation therapy is usually the first treatment.
  • Sipuleucel-T cell immunotherapy can be selected for the early stage of castration-resistant prostate cancer (CRPC), and drug therapies such as androgen inhibitors, androgen receptor antagonists, radiotherapy drugs for bone metastasis, and chemotherapy drugs acting on microtubules can be selected for the patients with metastatic castration-resistant prostate cancer (mCRPC) as appropriate.
  • CRPC castration-resistant prostate cancer
  • drug therapies such as androgen inhibitors, androgen receptor antagonists, radiotherapy drugs for bone metastasis, and chemotherapy drugs acting on microtubules
  • mCRPC metastatic castration-resistant prostate cancer
  • each of the therapies can only prolong survival by a few months, so it is necessary to seek effective therapies.
  • PSMA Prostate-specific membrane antigen
  • PSMA is a type II transmembrane glycoprotein expressed at high levels in prostate adenocarcinomas and at even higher levels in CRPCs. PSMA is one of the most clinically validated targets for diagnosis and treatment of prostate cancer. In addition to prostate epithelial cells, PSMA can also be expressed by nonprostate tissues, such as the small intestine, proximal renal tubules, and salivary glands, but at levels much lower than in the prostate tissue. PSMA is highly expressed in prostate cancer cells, particularly in metastatic diseases, hormone refractory diseases and high-grade lesions.
  • PSMA is also highly expressed in endothelial cells of neovasculature of all solid tumors, but not in normal vasculature, so it is a target for the treatment of solid tumors (Clin Cancer Res., 2010; 16(22): 5414-5423).
  • ADT and androgen receptor antagonist therapy can both upregulate the expression of PSMA (J Nucl Med, 2017; 58: 81-84), which provides the basis for targeted therapy in combination with traditional hormone therapy.
  • An antibody-drug conjugate links a monoclonal antibody or an antibody fragment to a biologically active cytotoxin via a linker compound, making full use of the binding specificity of the antibody to surface antigens of normal cells and tumor cells and the high efficiency of the cytotoxic substance, and also avoiding defects such as poor therapeutic effect of the antibody and serious toxic side effects of the toxic substance.
  • ADC can kill tumor cells more precisely and has a reduced effect on normal cells compared to conventional chemotherapeutic drugs.
  • ADC drugs have been used in nonclinical or clinical studies, such as Kadcyla, which is an ADC drug composed of Her2 -targeted trastuzumab and drug maytansinoid- 1 (DM1).
  • Kadcyla which is an ADC drug composed of Her2 -targeted trastuzumab and drug maytansinoid- 1 (DM1).
  • PSMA-targeted ADC drugs have been tested in clinical therapeutic studies. PSMA-ADC from Progenies Pharmaceuticals which reached phase II clinical testing before being discontinued, while MED 13726 from Medlmmune and MLN2704 from Millennium Pharmaceuticals were discontinued at the end of phase I clinical stage due to poor efficacy. Accordingly, there exists a need for improved ADC drugs targeting PSMA.
  • the present disclosure provides a conjugate having the structure of Formula (I):
  • Ab is an anti-prostate specific membrane antigen (PSMA) antibody, or an antigenbinding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:4, a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:5; and the VL comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:8;
  • PSMA anti-prostate specific membrane antigen
  • E is, at each occurrence, an exatecan payload
  • the conjugate has the following structure (II):
  • m is about 10 to about 20.
  • the conjugate has the structure of Formula (III): wherein each L 1 independently has one of the following structures: wherein * is the covalent bond to the Ab and ** is a covalent bond to the carbon marked with an arrow.
  • the anti-PSMA antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody, or de-immunized antibody.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2, wherein the amino acid sequences of HCDRs 1-3 and LCDRs 1-3 are unchanged.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO:2.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises: a human immunoglobulin G1 (IgGl) constant region.
  • the anti-PSMA antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.
  • the point of attachment of the linker to the anti-PSMA antibody or antigen binding fragment thereof is a cysteine residue of the anti-PSMA antibody or antigen binding fragment thereof.
  • the point of attachment is a cysteine residue at position 214 of SEQ ID NO: 12, a cysteine residue at position 218 of SEQ ID NO: 11, a cysteine residue at position 224 of SEQ ID NO: 11, and a cysteine residue at position 227 of SEQ ID NO: 11.
  • n is about 4 to about 16.
  • n is about 6 to about 8.
  • n is 6.
  • n 7.
  • n 8.
  • n 9.
  • n is 10.
  • the present disclosure provides a conjugate formed by reaction of a linker drug with an antibody, or an antigen binding fragment thereof, wherein the linker drug has the following structure: wherein m is about 0 to about 20; and the antibody, or antigen binding fragment thereof, is an anti-prostate specific membrane antigen (PSMA) antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:4, a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:5; and the VL comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:6, a light chain complementary
  • HCDR1 heavy chain complementary determining region 1
  • m is about 10 to about 20.
  • the linker drug has the following structure:
  • the anti-PSMA antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody, or de-immunized antibody.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2, wherein the amino acid sequences of HCDRsl-3 and LCDRsl-3 are unchanged.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO:2.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises a human IgGl constant region.
  • the anti-PSMA antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.
  • the one or more linker drugs forms a covalent bond with a cysteine residue at position 214 of SEQ ID NO: 12, a cysteine residue at position 218 of SEQ ID NO: 11, a cysteine residue at position 224 of SEQ ID NO: 11, and a cysteine residue at position 227 of SEQ ID NO:11.
  • the present disclosure provides a method of treating a subject having a PSMA-expressing cancer, comprising administering to a subject in need thereof an effective amount of any of the aforementioned conjugates or the aforementioned pharmaceutical composition.
  • the PSMA-expressing cancer is prostate cancer, salivary gland cancer, thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer.
  • the conjugate is administered at a dose of about 0.1 mg/kg to about 20 mg/kg.
  • the method results in complete inhibition of tumor growth for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days.
  • the conjugate is administered systemically, and in other embodiments the conjugate is administered intravenously.
  • FIG. 1 is a schematic representation of an embodiment of the antibody drug conjugate molecule of the present disclosure (ALD101).
  • the HUJ591 antibody is conjugated to the linker payload.
  • the chemical structure of the linker payload is shown on the right side.
  • LC refers to the light chain (e.g., SEQ ID NO: 12);
  • HC refers to the heavy chain (e.g., SEQ ID NO: 11).
  • FIGS. 9A-9D show in vitro cytotoxicity assays in prostate cancer cell lines treated with exatecan.
  • Cell viability assay in LNCaP (FIG. 9A), LNCaP-abl (FIG. 9B), 22Rvl (FIG. 9C), and PC3 (FIG. 9D) cell lines after exatecan treatment for 4 days, using a sulforhodamine B (SRB) assay.
  • SRB sulforhodamine B
  • FIGS. 10A-10D show ALD101 cytotoxicity in prostate cancer cell lines.
  • Cell viability assay in LNCaP (FIG. 10A), LNCaP-abl (FIG. 10B), 22Rvl (FIG. 10C), and PC3 (FIG. 10D) cell lines after treatment with ALD101 (closed square), nonbinding isotype control ADC (ADC control; open square), or unconjugated HUJ591 mAb (naked mAb; closed circle) for 4 days, using an SRB assay.
  • Tumor-bearing mice were treated when tumors reached a volume of approximately 125 mm 3 with vehicle (black circle), 10 mg/kg nonbinding isotype control ADC (ADC Ctrl; triangle), 3 mg/kg ALD101 (diamond), or 10 mg/kg ALD101 (square). A single dose of each treatment was administered, and mice were monitored for tumor growth 3 times/week. Data are presented as mean ⁇ SEM.
  • FIG. 13 shows mouse weight after ALD101 treatment in an LNCaP-abl CDX model. Mouse weight was monitored to evaluate general health status after treatment with vehicle (filled circle), 10 mg/kg nonbinding control ADC (triangle), 3 mg/kg ALD101 (diamond), or 10 mg/kg ALD101 (square). Data are shown as mean ⁇ SD.
  • FIG. 15 shows in vivo antitumor activity of ALD101 in an LNCaP-abl CDX mouse model comparing 10 mg/kg vs 15 mg/kg.
  • Tumor-bearing mice were treated when the tumor reached a volume of approximately 175 mm 3 with vehicle (filled circle), 10 mg/kg ALD101 (square), or 15 mg/kg ALD101 (triangle).
  • a single dose of each treatment was administered, and mice were monitored for tumor growth 3 times/week. Data are presented as mean ⁇ SEM.
  • FIG. 16 shows mouse weight after ALD101 treatment in an LNCaP-abl CDX model. Mouse weight was monitored to evaluate general health status after treatment with a single dose of vehicle (circle), 10 mg/kg ALD101 (square), or 15 mg/kg ALD101 (light triangle). Data are shown as mean ⁇ SD.
  • FIGS. 17A-17D show distribution of exatecan in different body compartments after treatment with 3 and 10 mg/kg ALD101.
  • Exatecan was quantified in biological matrices from LNCaP-abl CDX mice treated with 3 or 10 mg/kg ALD101 (black, 4 mice per group) or nonbinding isotype control ADC (ADC CTRL, white; 3 mice per group), and sacrificed 24 hours later.
  • FIG. 17B plasma (ng/mL plasma),
  • FIG. 17C liver tissue (ng/g tissue).
  • FIG. 17D Ratio of exatecan (per mass tumor versus mass of exatecan/mL plasma).
  • FIG. 20 shows tumor growth curves for each pre-treatment tumor size group in an LNCaP-abl CDX model treated with a single dose of ALD101 10 mg/kg according to Example 5.
  • FIG. 21 shows an in vivo experimental plan for antitumor efficacy evaluation of 3 doses of ALD101 (7.5 mg/kg) in an LNCaP-abl CDX model treated according to Example 6.
  • FIG. 22 shows in vivo antitumor activity in mice treated with 3 doses of ALD101 (7.5 mg/kg) or vehicle in an LNCaP-abl CDX model treated according to Example 6. Tumorbearing mice were treated when the tumor reached a volume of approximately 300 mm 3 .
  • FIG. 23 shows mouse weight after 3 doses of ALD101 (7.5 mg/kg) or vehicle treatment in an LNCaP-abl CDX model treated according to Example 6.
  • FIG. 26 shows mouse weight after vehicle or single or repeated doses of ALD101 (10, 20 or 10x2 mg/kg) in an LNCaP-abl CDX model treated according to Example 7.
  • FIG. 27 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (10x2, 15x2 or 20 mg/kg) in an LNCaP-abl CDX model according to Example 8.
  • FIG. 28 shows in vivo antitumor activity of single or repeated doses of ALD101 (10x2, 15x2 or 20 mg/kg) in an LNCaP-abl CDX mouse model treated according to Example 8. Tumor-bearing mice were treated when the tumor reached a volume of approximately 300 mm 3 .
  • FIG. 29 shows mouse weight after vehicle or single or repeated doses of ALD101 (10x2, 15x2 or 20 mg/kg) in an LNCaP-abl CDX model treated according to Example 8.
  • FIG. 30 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (7.5 mg/kg xl x2 x3 versus 20 mg/kg) in an LNCaP-abl CDX model treated according to Example 9.
  • FIG. 31 shows in vivo antitumor activity of single or repeated doses of ALD101 (7.5 mg/kg xl x2 x3 versus 20 mg/kg) in an LNCaP-abl CDX model treated according to Example 9. Tumor-bearing mice were treated when the tumor reached a volume of approximately 200 mm 3 .
  • FIG. 32 shows mouse weight after vehicle or single or repeated doses of ALD101 (7.5 mg/kg xl x2 x3 versus 20 mg/kg) in an LNCaP-abl CDX model according to Example 9.
  • FIG. 74 shows total ADC and total IgG in plasma after a single administration of ALD101 at 10 mg/kg in 22Rvl CDX mice according to Example 22.
  • any number range of the disclosure relating to any physical feature, such as polymer subunits, size, or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated.
  • numerical ranges are inclusive of their recited endpoints, unless specifically stated otherwise.
  • a binding domain and a fusion protein thereof “specifically binds” a target if it binds the target with an affinity or Ka (z.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M’ 1 , while not significantly binding other components present in a test sample.
  • Binding domains (or fusion proteins thereof) may be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof).
  • “High affinity” binding domains refer to those binding domains with a Ka of at least 10 8 M’ 1 , at least 10 9 M’ 1 , at least IO 10 M’ 1 , at least 10 11 M’ 1 , at least 10 12 M’ 1 , or at least 10 13 M’ 1 , preferably at least 10 8 M' 1 or at least 10 9 M' 1 .
  • “Low affinity” binding domains refer to those binding domains with a Ka of up to 10 8 M’ 1 , up to 10 7 M’ 1 , up to 10 6 M’ 1 , up to 10 5 M' 1 .
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10' 5 M to 10’ 13 M).
  • binding domain polypeptides and fusion proteins can be readily determined using conventional techniques (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51 :660, 1949; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
  • identity refers to the similarity between a DNA, RNA, nucleotide, amino acid, or protein sequence to another DNA, RNA, nucleotide, amino acid, or protein sequence. Identity can be expressed in terms of a percentage of sequence identity of a first sequence to a second sequence. Percent (%) sequence identity with respect to a reference DNA sequence can be the percentage of DNA nucleotides in a candidate sequence that are identical with the DNA nucleotides in the reference DNA sequence after aligning the sequences.
  • Percent (%) sequence identity with respect to a reference amino acid sequence can be the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid 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.
  • the percent sequence identity values for sequences provided herein can be generated using the NCBI BLAST 2.0 software as defined by Altschul et al., “Gapped BLAST and PSLBLAST: a new generation of protein database search programs,” Nucleic Acids Res. 1997, 25, 3389-3402, with the parameters set to default values.
  • Ab is an anti-PSMA antibody or antigen binding fragment thereof
  • anti-PSMA antibodies also referred to as PSMA binding antibodies
  • antigen-binding fragments thereof that specifically bind to PSMA for use in conjugates of anti-PSMA antibodies or antigen binding fragments thereof and cytotoxic agents, such as exatecan, also referred to herein as anti-PSMA conjugates.
  • the anti-PSMA conjugates of the present disclosure reduce the number of PSMA+ cancer cells in a subject.
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL),
  • VH region comprises a heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO:4, and a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 5; and
  • HCDR1 heavy chain complementary determining region 1
  • HCDR2 heavy chain complementary determining region 2
  • HCDR3 heavy chain complementary determining region 3
  • VL region comprises a light chain complementary determining region 1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO:6, a light chain complementary determining region 2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 8, wherein the antibody or antigen-binding fragment thereof specifically binds to PSMA.
  • LCDR1 light chain complementary determining region 1
  • LCDR2 light chain complementary determining region 2
  • LCDR3 light chain complementary determining region 3
  • the anti-PSMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PSMA antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2, provided that the amino acid sequences of the HCDRs 1-3 and LCDRs 1-3 are unchanged.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PSMA antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:2.
  • VH heavy chain variable region
  • VL light chain variable region
  • an anti-PSMA antibody or antigen binding fragment thereof may be identified by its heavy and/or light chain CDRs using any one of the following methods: Kabat, Chothia, AbM, Contact, IMGT, and/or Aho.
  • an anti- PSMA antibody or antigen-binding fragment thereof may be identified by CDRs, variable regions, and heavy and light chains provided in Table 1. The CDRs shown in Table 1 are as defined by Kabat numbering.
  • an antibody or antigen-binding fragment thereof comprises two light chain polypeptides (light chains) and two heavy chain polypeptides (heavy chains), held together covalently by disulfide linkages.
  • the heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region typically comprises three domains, CHI, CH2, and CH3.
  • Nonlimiting exemplary heavy chain constant regions include human IgGl, human IgG2, human IgG3, and human IgG4 constant regions.
  • an antibody of the disclosure comprises an IgGl constant region.
  • An exemplary heavy chain constant region includes human IgGl heavy chain constant region (SEQ ID NO: 15).
  • the light chain comprises a light chain variable region (VL) and a light chain constant region.
  • VL light chain variable region
  • Nonlimiting exemplary light chain constant regions include kappa and lambda constant regions.
  • a nonlimiting exemplary human kappa constant region is shown in SEQ ID NO: 14.
  • Fc region or domain may interact with different types of FcRs.
  • the different types of FcRs may include, for example, FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcaRI, FcpR, FcaRI, FcsRII, and FcRn.
  • FcRs may be located on the membrane of certain immune cells including, for example, B lymphocytes, natural killer cells, macrophages, neutrophils, follicular dendritic cells, eosinophils, basophils, platelets, and mast cells.
  • the FcR may initiate functions including, for example, clearance of an antigenantibody complex via receptor-mediated endocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), trogocytosis, trogoptosis, and ligand-triggered transmission of signals across the plasma membrane that can result in alterations in secretion, exocytosis, and cellular metabolism.
  • FcRs may deliver signals when FcRs are aggregated by antibodies and multivalent antigens at the cell surface.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • SRC family tyrosine kinases may sequentially activate SRC family tyrosine kinases and SYK family tyrosine kinases.
  • ITAM comprises a twice-repeated YxxL sequence flanking seven variable residues.
  • the SRC and SYK kinases may connect the transduced signals with common activation pathways.
  • an Fc region or domain can exhibit reduced binding affinity to one or more Fc receptors. In some embodiments, an Fc region or domain can exhibit reduced binding affinity to one or more Fey receptors. In some embodiments, an Fc region or domain can exhibit reduced binding affinity to FcRn receptors. In some embodiments, an Fc region or domain can exhibit reduced binding affinity to Fey and FcRn receptors. In some embodiments, an Fc domain is an Fc null domain or region. As used throughout the disclosure, an “Fc null” refers to a domain that exhibits weak to no binding to any of the Fey receptors. In some embodiments, an Fc null domain or region exhibits a reduction in binding affinity (e.g., increase in Kd) to Fey receptors of at least about 1000-fold.
  • the Fc region or domain may have one or more, two or more, three or more, or four or more, or up to five amino acid substitutions that decrease binding of the Fc region or domain to an Fc receptor.
  • an Fc region or domain exhibits decreased binding to FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD 16a), FcyRIIIB (CD 16b), or any combination thereof.
  • an Fc region or domain may comprise one or more amino acid substitutions that has the effect of reducing the affinity of the Fc domain or region to an Fc receptor.
  • the Fc region or domain can comprise a sequence of the IgGl isoform that has been modified from the wild-type IgGl sequence.
  • a modification can comprise a substitution at more than one amino acid residue, such as at 5 different amino acid residues including L235V/F243L/R292P/Y300L/P396L (IgGIVLPLL) according to the EU index of Kabat numbering.
  • a modification can comprise a substitution at more than one amino acid residues, such as at two different amino acid residues including S239D/I332E (IgGIDE) according to the EU index of Kabat numbering.
  • a modification can comprise a substitution at more than one amino acid residue, such as at three different amino acid residues including S298A/E333A/K334A (IgGl AAA) according to the EU index of Kabat numbering.
  • IgGl AAA S298A/E333A/K334A
  • a non- limiting exemplary human IgGl heavy chain constant regions is shown in SEQ ID NO: 15.
  • an antibody of the disclosure comprises a mouse IgG2a heavy chain constant region.
  • an antibody of the disclosure comprises a rat IgG2b heavy chain constant region.
  • An antibody or Fc domain may be modified to acquire or improve at least one constant region-mediated biological effector function relative to an unmodified antibody or Fc domain, e.g., to enhance FcyR interactions.
  • a modification can increase CD32b binding (and support transdelivery in a PBMC assay) comprises a substitution at S267L and E329F (IgGILF, also known as SELF double mutant) according to the EU index of Kabat numbering.
  • an antibody with a constant region that binds to FcyRIIA, FcyRIIB and/or FcyRIIIA with greater affinity than the corresponding wild type constant region may be produced according to the methods described of the disclosure.
  • An Fc domain that binds to FcyRIIA, FcyRIIB and/or FcyRIIIA with greater affinity than the corresponding wild type Fc domain may be produced according to the methods of the disclosure.
  • an Fc region or domain found in an anti-PSMA antibody of the disclosure is capable of mediating one or more of these effector functions, or lacks one or more or all of these activities or have one or more of the effector activities increased by way of, for example, one or more mutations as compared to the unmodified Fc region or domain.
  • the one or more linker drugs forms a covalent bond with a cysteine residue at one or more of the following positions: position 214 of the antibody light chain, position 218 of the antibody heavy chain, position 224 of the antibody heavy chain, position 227 of the antibody heavy chain, or any combination thereof.
  • the positions of the cysteine residues in the antibody light chain and antibody heavy chain may be made in reference to SEQ ID NO: 12 and SEQ ID NO: 11, respectively.
  • the one or more linker drugs forms a covalent bond with a cysteine residue at position 214 of the antibody light chain, position 218 of the antibody heavy chain, position 224 of the antibody heavy chain, and position 227 of the antibody heavy chain.
  • m is about 10 to about 20. In a further embodiment, m is 11.
  • the total concentration of the buffer in the aqueous formulation is between 10 mM and 40 mM. In some embodiments, the total concentration of the buffer in the aqueous formulation is between 15 mM and 30 mM. In some embodiments, the total concentration of the buffer in the aqueous formulation is between 15 mM and 25 mM. In some embodiments, the total concentration of the buffer in the aqueous formulation is 20 mM or about 20 mM.
  • the aqueous formulation comprises at least one lyoprotectant.
  • the at least one lyoprotectant is selected from sucrose, arginine, glycine, sorbitol, glycerol, trehalose, dextrose, alpha-cyclodextrin, hydroxypropyl beta-cyclodextrin, hydroxypropyl y-cyclodextrin, proline, methionine, albumin, mannitol, maltose, dextran, and combinations thereof.
  • the lyoprotectant is sucrose.
  • the total concentration of lyoprotectant in the aqueous formulation is 3-12%, such as 5-12%, 6-10%, 5-9%, 7-9%, or 8%.
  • the aqueous formulation comprises at least one surfactant.
  • Exemplary surfactants include polysorbate 80, polysorbate 20, poloxamer 88, and combinations thereof.
  • the aqueous formulation comprises polysorbate 80.
  • the total concentration of the at least one surfactant is 0.01%-0.1%, such as 0.01%-0.05%, 0.01%-0.08%, or 0.01%-0.06%, 0.01%-0.04%, 0.01%-0.03%, or 0.02%.
  • compositions may have conjugates of this disclosure with an average ratio of the drug (exatecan) to anti-PSMA antibody or antigen binding fragment thereof (referred to herein as a drug-to-antibody ratio, or DAR) that ranges from 1 to about 20, from about 1 to about 16, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 5, from 1 to about 3, from about 2 to about 16, from about 2 to about 10, from 2 to about 8, from 2 to about 6, from 2 to about 5, from 2 to about 4, from about 3 to about 16, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, or from about 3 to about 5, from about 4 to about 16, from about 4 to about 10, from about 4 to about 8, from about 5 to about 10, from about 5 to about 8, from about 5 to about 8, from about 6 to about 10, from about 6 to about 8, wherein the drug is exatecan.
  • DAR drug-to-antibody ratio
  • the average ratio of the exatecan to anti- PSMA antibody or antigen binding fragment thereof of conjugates in a pharmaceutical formulation may range from 1 to 20, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 5, from 1 to 3, from 2 to 8, from 2 to 6, from 2 to 5, from 2 to 4, from 3 to 8, from 3 to 6, from 3 to 5, from 4 to 8, or from 6 to 8, wherein the drug is exatecan.
  • the average ratio of the conjugate is 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, 6 or about 6, 7 or about 7, or 8 or about 8.
  • the present disclosure provides methods of treating or preventing diseases or conditions (e.g., PSMA expressing cancers, also referred to PSMA+ cancers) comprising administering to a subject in need thereof an effective amount of anti-PSMA conjugates or compositions thereof disclosed herein.
  • diseases or conditions e.g., PSMA expressing cancers, also referred to PSMA+ cancers
  • present disclosure provides use of anti- PSMA conjugates or compositions thereof disclosed herein in the manufacture of a medicament for treating or preventing diseases or conditions, such as a PSMA expressing cancer.
  • a subject in need of treatment has been diagnosed with the disease or condition.
  • the subject has been treated with another therapy.
  • the subject is resistant to another therapy or has relapsed following administration of another therapy, and therefore, is in need of treatment by providing or administrating to the subject one or more of an anti-PSMA conjugate or a composition thereof of this disclosure.
  • a therapeutically effective amount of one or more of an anti-PSMA conjugate or composition thereof of this disclosure is administered to a subject in need thereof.
  • therapeutically effective amounts of the anti-PSMA conjugates and pharmaceutical compositions thereof can be administered to a subject in need thereof, often for treating or preventing a condition or progression thereof.
  • the anti-PSMA conjugates and pharmaceutical compositions thereof of this disclosure can affect the physiology of the subject, such as the immune system, an inflammatory response, or other physiologic affect.
  • a therapeutically effective amount can vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.
  • an effective amount or “effective dose” refers to a quantity of a binding protein conjugate or composition thereof sufficient to achieve a desired (e.g., beneficial) effect in a subject being treated with that compound, conjugate, or composition thereof, such as an amount sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner, delaying worsening of a progressive disease in a statistically significant manner, or preventing onset of additional associated symptoms or diseases in a statistically significant manner, or any combination thereof.
  • an effective amount of a binding protein conjugate or composition thereof is an amount sufficient to inhibit or treat the disease with minimal to no toxicity in the subject, excluding the presence of one or more adverse side effects.
  • An effective amount or dose can be administered one or more times over a given period of time.
  • An effective amount or dose can depend on the purpose of the treatment and can be ascertainable by one skilled in the art based on a subject’s needs.
  • an effective amount or dose refers to that ingredient alone.
  • an effective amount or dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously.
  • a prophylactic treatment meant to “prevent” a disease or condition is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs, for the purpose of decreasing the risk of developing pathology or further advancement of the early disease. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
  • a prophylactic treatment can mean preventing recurrence of a disease or condition in a patient that has previously been treated for the disease or condition, e.g., by preventing relapse or recurrence of cancer.
  • PSMA expressing cancers can be classified as having a low, intermediate or heterogeneous, or high expression level of PSMA.
  • a low expression level refers to a cancer having less than 1 x 10 3 PSMA ligands per cell; an intermediate or heterogeneous expression level refers to a cancer having from 1 x 10 3 to 1 x 10 4 PSMA ligands per cell; a high expression level refers to a cancer having greater than 1 x 10 4 PSMA ligands per cell.
  • Expression levels for purposes of classifying the cancers as either low, intermediate or heterogeneous, or high PSMA expression levels is determined by flow cytometry methods known to those of ordinary skill in the art and demonstrated herein in the examples.
  • PSMA expressing cancers that may be treated with the conjugates and compositions of the disclosure include prostate cancer, salivary gland cancer, thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer.
  • the PSMA expressing cancer is metastatic.
  • the PSMA expressing cancer is metastatic prostate cancer.
  • the PSMA expressing cancer is metastatic castrate resistant prostate cancer.
  • the PSMA-expressing cancer expresses an intermediate level of PSMA. In other embodiments, the PSMA-expressing cancer expresses a high level of PSMA. In other embodiments, the PSMA-expressing cancer expresses a heterogeneous level of PSMA. In various embodiments, the tumor to plasma ratio of free exatecan in the subject is within 30% of the tumor to plasma ratio of free exatecan in a subject having a high PSMA - expressing cancer, preferably wherein the tumor to plasma ratio of free exatecan is within 25%, 20%, 15%, 10% or 5%.
  • the tumor to plasma ratio is higher in the subject having a PSMA-expressing cancer that expresses an intermediate or heterogeneous level of PSMA as compared to the tumor to plasma ratio in a subject having a PSMA-expressing cancer that expresses high level of PSMA.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • intravenous administration and “administered intravenously” as used herein refer to injection or infusion of a conjugate into a vein of a subject.
  • Conjugates and compositions of the disclosure may be administered to a subject once every 1, 2, 3, or 4 weeks. Conjugates and compositions of the disclosure may be administered to a subject once every 1-4 weeks, every 1-3 weeks, or every 1-2 weeks. Conjugates and compositions of the disclosure may be administered to a subject once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • each dose or the total dose per treatment cycle may comprise between about 1 mg/kg and about 15 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise between 1 mg/kg and 10 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise between about 1 mg/kg and about 10 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
  • each dose or the total dose per treatment cycle may comprise 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mg/kg or any number of mg/kg in between.
  • each dose or the total dose per treatment cycle may comprise about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, about 30 mg/kg or any number of mg/kg in between.
  • the treatment cycle comprises one or more treatment administrations and one or more periods of observation.
  • the treatment cycle comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In some embodiments, the treatment cycle comprises at least 1, 2, 3, or 4 weeks. In some embodiments, the treatment cycle comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • Conjugates and compositions of this disclosure may be administered to a subject in combination with an additional therapy or therapeutic agent.
  • an additional therapy or therapeutic agent comprises surgery, a hormone therapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, a molecularly targeted therapeutic agent, thermotherapy, radiation therapy, or a vaccine.
  • Hormone therapy also referred to as androgen suppression therapy, for prostate cancer aims to reduce the levels of androgens in the body or stop them from fueling prostate cancer growth.
  • hormone therapy comprises androgen deprivation therapy, anti -androgen therapy (also referred to androgen receptor antagonists), and estrogen therapy. Androgen deprivation therapy targets production of androgens.
  • androgen deprivation therapy comprises a luteinizing hormone-releasing hormone (LHRH) agonist, such as leuprolide, goserelin, triptorelin, or leuprolide mesylate; a LHRH antagonist, such as degarelix or relugolix; a CYP17 inhibitor, such as abiraterone; or ketoconazole.
  • LHRH luteinizing hormone-releasing hormone
  • anti -androgen therapy comprises bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, proxalutamide, cimetidine, or topilutamide.
  • Chemotherapeutic agents may include erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FTJ (5 -fluorouracil), 5-FU (5 -fluorouracil) leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafamib (SCH 66336, ZokinvyTM, Eiger BioP
  • Immunotherapeutic agents include immunostimulatory agents, immune checkpoint inhibitors, antibody therapy, and cellular immunotherapy.
  • Immunotherapeutic agents include naturally occurring molecules or cells (e.g., monoclonal antibodies, TCRs) and non- naturally occurring molecules or cells, such as recombinant or fusion molecules (e.g., bispecific T cell engager).
  • immunostimulatory agents include cytokines, such as IL-2, IL-12, IL-15, IFNa, IFNy, TNFa; chemokines, such as CCL21; and immunostimulatory oligonucleotides.
  • immune checkpoint inhibitors include inhibitors targeting PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, PVRIG/CD112R, or any combination thereof.
  • an immune checkpoint inhibitor may be an antibody, a peptide, an RNAi agent, or a small molecule.
  • An antibody specific for CTLA-4 may be ipilimumab or tremelimumab.
  • An antibody specific for PD-1 may be pidilizumab, nivolumab, or pembrolizumab.
  • An antibody specific for PD-L1 may be durvalumab, atezolizumab, or avelumab.
  • Examples of cellular immunotherapy include T cell receptors (TCRs), tumor infiltrating lymphocytes, and chimeric antigen receptors (CARs).
  • a cancer vaccine may be a non-antigen specific vaccine (e.g., whole cell vaccine) or an antigen-specific vaccine, such as a protein vaccine, peptide vaccine, protein vaccine, cancer-associated membrane carbohydrate vaccine, mRNA vaccine, nucleic acid vaccine, or antigen-loaded dendritic cell vaccine (e.g., sipuleucel-T).
  • Radiation therapy includes external beam radiation therapy e.g., conventional external beam radiation therapy, stereotactic radiation, 3 -dimensional conformal radiation therapy, intensity-modulated radiation therapy, volumetric modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, systemic radioisotope therapy, intraoperative radiotherapy, or any combination thereof.
  • the conjugates and compositions of this disclosure may be administered to a subject in combination with an agent targeting other prostate cancer targets, for example, STEAP1, or B7H3.
  • TGI tumor growth inhibition
  • the present disclosure provides a method for treating a subject having a PSMA expressing cancer, comprising administering to a subject in need thereof a predetermined amount of the conjugate of Formula (I), wherein the predetermined amount is administered to the subject in two or more doses within a 30-day period (i.e., the predetermined amount is split into two or more doses, which are administered separately within the 30-day period).
  • the predetermined amount is administered to the subject in two doses within the 30-day period
  • the predetermined amount is administered to the subject in three doses within the 30-day period.
  • the predetermined amount is administered to the subject in more than three doses within the 30-day period.
  • the two or more doses are administered at a 7 to 15-day interval.
  • the two or more doses are administered at a 7-day interval, 8-day interval, 9-day interval or 10-day interval.
  • the two or more doses are administered within a 20-day period.
  • the predetermined amount is administered in three doses within a 20-day period.
  • the administering results in an exatecan tumor to plasma ratio in the subject of at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100: 1, at least 110:1, at least 120: 1, at least 130: 1, at least 140: 1 or at least 150: 1.
  • a period of mean tumor growth inhibition (TGI) in the subject is extended compared to administering the approximately predetermined amount in a single dose.
  • TGI tumor growth inhibition
  • the TGI is extended at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, or at least 400%.
  • the method results in tumor growth inhibition (TGI) in the subject for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days.
  • TGI tumor growth inhibition
  • the TGI is 60%, 70%, 80%, 90% or greater.
  • the method results in complete inhibition of tumor growth for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days.
  • the subject has a PSMA expressing cancer with a tumor volume of at least 125 mm 3 , at least 175 mm 3 , at least 200 mm 3 , at least 250 mm 3 , at least 300 mm 3 , at least 350 mm 3 , at least 400 mm 3 , at least 450 mm 3 , at least 500 mm 3 , at least 550 mm 3 , at least 600 mm 3 or at least 650 mm 3 prior to administering the two or more doses.
  • the conjugate is administered systemically, and in other embodiments the conjugate is administered intravenously.
  • reaction solution was purified directly by reverse phase liquid chromatography (40 g C18 column, eluting with 0- 70% acetonitrile in water with 0.01% TFA over 15 min) to yield compound 101-8 (270 mg, 0.135 mmol, 63.48%) as a white solid.
  • LCMS, ESI m/z 666.6 (M/3+H) , 999.2 (M/2+H) + .
  • ALD101 was purified with a Pellicon 3, 30 kD cassette with Ultracel® membrane (0.11 m 2 ) and then filtered with a 0.2 pm sterile filter. This yielded a conjugate with 8 moles of LD101 per mole of antibody.
  • the C-terminal sequence of the anti-PSMA heavy chain was confirmed as SLSLSPGK, with the loss of the C-terminal lysine as a modification typically seen in IgGl monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Low ratio unmodified peptide SLSLSPGK was also observed.
  • Flow cytometry was performed on tumor tissue (tumor tissue from CDX mice described above) and PC cell lines (cells at a confluency of 85% were removed from the flask with 1 mM EDTA in PBS). Single-cell suspensions of tumor cells were processed for analysis using PBS with 1% FBS, 2 mmol/L EDTA, and fluorochrome-conjugated monoclonal antibodies (mAbs) against PSMA (phycoerythrin-conjugated anti-human PSMA, LNI-17, BioLegend, #342504) or unconjugated PSMA (HUJ591, Wuxi).
  • PSMA fluorochrome-conjugated monoclonal antibodies
  • PC cell lines were seeded (l-2xl0 4 cells) on glass coverslips treated with polylysine (Sigma) in 24-well plates and incubated for 24 hours at 37 °C in 5% CO2 with medium (according to the cell line).
  • Cells were fixed with 4% paraformaldehyde for 10 min at RT and stained with anti-human PSMA mAb (LNI-17, BioLegend or HUJ591, Wuxi) followed by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Life Technologies). Immunofluorescence imaging of cells was performed using a 20-fold magnification 1.25 NA with an inverted CSLM Leica Stellaris 5 confocal microscope.
  • Cells were seeded in a 96-well plate at an appropriate density according to the cell line (typically 1000 to 3000 cells per well in 100 pL of appropriate culture media). After an overnight incubation, a concentration series of exatecan (cat# HY- 13631 A, Lucerna AG), ALD101, a control nonbinding ADC, or unconjugated antibody (HUJ591, Wuxi) was added. The maximum concentration tested was 200 nM for each compound. Exatecan alone was diluted 1 :5 by serial dilution, and unconjugated mAb, ALD101, and control nonbinding ADC were diluted 1 : 10 by serial dilution.
  • Cell viability was evaluated after 4 days using an SRB assay for cell density determination, based on the measurement of cellular protein content.
  • Cell monolayers were fixed with 10% (w/v) trichloroacetic acid, stained with 0.057% (w/v) SRB sodium salt, and the stain reconstituted with 10 mM Tris-base buffer pH 10.
  • Absorbance was measured at 510 nm using a microplate reader (Cytation 5 Imaging multi -Readers, Agilent Technologies). The IC50 values were compared with untreated cells or cells treated with controls, and were determined using inhibition dose-response curve fitting (GraphPad Prism 9). Target expression in each cell line was measured by flow cytometry as described above.
  • Tumors were harvested and fixed in 10% neutral -buffered formalin (cat# 5701, Thermo Scientific) overnight and embedded in paraffin according to standard protocols. Slides were prepared using consecutive sections. For antigen retrieval, slides were incubated for 60 min in citrate buffer at pH 6 at 98 °C. Slides were incubated with a mouse mAb against PSMA (cat# M3620, Dako) at the dilution of 1 :400. PSMA detection was performed using the Refine Detection DAB Kit (Leica). Protein blocking was performed using Protein-Block solution (cat# X0909, DAKO Agilent Technologies) for 10 mins at RT.
  • Tumor tissues 25-30 mg or cell pellets from cell lines were lysed with RIP A buffer supplemented with cocktail phosphatase inhibitors (cat# 4906845001, Roche) and protease inhibitors (cat# 5892953001, Roche).
  • Whole protein lysate was separated on 8-12% SDS-polyacrylamide gels and transferred onto a PVDF membrane (cat# 88518, Thermo Fisher Scientific). The membranes were incubated overnight at 4 °C with primary antibodies, anti- GAPDH (cat# sc-47724, Santa Cruz) and anti-PSMA (cat# OTI3H5, Bio-Techne).
  • Detection was done using secondary antibodies (anti -rabbit IgG HRP, cat# W401B and anti -mouse IgG HRP, cat# W402B, Promega).
  • the protein bands were visualized using the western bright quantum reagent (cat# K-12042-D20, Advansta) and quantified using the Fusion Solo IV LBR system (Witec AG).
  • a CDX model was generated using male mice (NRG mouse model) aged 6 to 8 weeks. Mice were castrated following a specific SOP, and 7 to 10 days later, a single preparation of 2xl0 6 LNCaP-abl cells was injected subcutaneously into the mouse flank using 1 : 1 Matrigel® Matrix (cat# 354234, Corning) with PBS. The single cell suspension was kept on ice until injection. Cells at low passage number in culture (not more than 20 passages in culture) were used, after Mycoplasma screening. Sporadic cases of renal masses contiguous with the subcutaneous tumor at the injection site were observed.
  • tumor volumes were measured three times per week in two dimensions using a caliper. Tumor growth was recorded using a digital caliper, and the tumor volume (TV), expressed in mm 3 , was calculated by the formula: length x (width) 2 /2. Randomization
  • Randomization was performed for antitumor evaluation when the mean tumor volume reached approximately 125-175 mm 3 .
  • the mice were randomized according to their tumor volume to ensure that the tumor volume in different groups was similar.
  • the date of randomization was denoted as Day 0 and corresponds to the treatment administration day per study design.
  • Xenograft monitoring started 1 week after PC cell injection and was performed three times per week (FIG. 3). Tumor volumes were measured three times per week. As soon as tumors reached a volume of between 125 to 175 mm 3 (approximately 2 weeks after injection), the mice were randomly assigned to the treatment groups or vehicle and treated as described in Table 5 and Table 6.
  • Table 5 ADC treatment plan to evaluate antitumor efficacy in an LNCaP-abl CDX model
  • Table 6 ADC treatment plan to evaluate antitumor efficacy at a higher ADC dose in an LNCaP-abl CDX model
  • FIG. 7 Comparison of fluorochrome-conjugated antibody and unconjugated anti-human PSMA HUJ591 by flow cytometry is shown in FIG. 7 and immunofluorescence in FIG. 8.
  • FIG. 9 In vitro cytotoxicity assays with exatecan and ALD101 in PC cell lines are shown in FIG. 9 and FIG.
  • Xenograft monitoring started 1 week after injection of LNCaP-abl PC cells and was performed 3 times per week. Tumor volumes were measured 3 times per week. As soon as tumors reached a volume of between 200 to 250 mm 3 (approximately 2 weeks after injection), the mice were randomly assigned to the treatment groups or vehicle, and treated as described in Table 12.
  • Ion transfer tube temperature 325 °C
  • Analytical data were acquired using Chromelion 7.3 chromatography data system (CDS) software (ThermoFisher Scientific). Peak area ratios from calibration samples (area of analyte peak divided by area of IS peak) were used to calculate the intercept and the slope of the calibration curve by the weighted least squares method. Calibration curve parameters were calculated using an appropriate weighing factor (l/x2). The goodness of fit of the linear regression was assessed using the coefficient of determination R2 and the back-calculated concentrations of the individual calibration standards.
  • CDS Chromelion 7.3 chromatography data system
  • mice Number of mice (LNCaP-abl CDX) with evaluation of antitumor efficacy following a single dose of ALD101 10 mg/kg
  • xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. Following these initial daily observations, CDX tumor growth was monitored three times per week until the tumor reached a maximum volume of 800-1500 mm 3 or until the maximum observation period specified by the animal license was reached, at which point the animals were sacrificed. Tumor tissue was then collected from each mouse for formalin-fixed paraffin-embedded (FFPE) blocks for immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry.
  • FFPE formalin-fixed paraffin-embedded
  • Tumor volume data over time from mice treated with ALD101 10 mg/kg in the 4 studies were pooled and analyzed in 3 groups according to the size of the tumor immediately prior to treatment: below 250 mm 3 , between 250 and 350 mm 3 and above 350 mm 3 .
  • Data were analyzed for tumor volume measurements for mice with tumor measurements through to 40 days after administration of ALD101 10 mg/kg on Day 0; note that for mice treated in study 4 that received a second dose of ALD101 10 mg/kg on Day 18, only tumor volume data collected up to Day 18 before the second injection were included in the current analysis.
  • the number of mice included per analysis group is presented in Table 16. Note also that animals presenting signs of tumor ulcerations/necrosis were sacrificed and were excluded from grouped analyses.
  • FIG. 20 shows tumor growth curves for each pre-treatment tumor size group in CDX NRG mice, from treatment start (Day 0 corresponding to randomization and ALD101 administration) until Day 40.
  • TGI tumor growth inhibition
  • Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. Tumor volumes were measured three times per week. When tumors reached a volume of approximately 300 mm 3 (approximately 2-3 weeks after injection), the mice were randomly assigned to the treatment group or vehicle and treated as described in Table 17.
  • Table 17 ADC Treatment Plan to Evaluate Antitumor Efficacy with Repeated ADC Doses in an LNCaP-abl CDX Model.
  • xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm 3 or after 82 days, and animals were sacrificed. Tumor tissue was then collected from each mouse for formalin- fixed paraffin-embedded (FFPE) blocks for future immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry.
  • FFPE formalin- fixed paraffin-embedded
  • Tumor growth curves (presented as mean ⁇ SEM) and mouse body weight (presented as mean ⁇ SD) for each treatment group from treatment start until study termination were measured are shown in FIG. 22 and FIG. 23, respectively.
  • ALD101 showed antitumor effects in vivo in a castrated LNCaP-abl CDX immunodeficient NRG model, with a mean tumor volume of 300 mm 3 (range -200-440 mm 3 ) at the time of treatment. Major and early tumor inhibition was seen in these mice with large tumors treated with three doses of ALD101 7.5 mg/kg (z.e., lower than the standard dose of 10 mg/kg), with mean TGI >90% lasting 56 days. TGI was durable with complete inhibition of tumor growth lasting for 43 days after the third injection of ALD101 7.5 mg/kg. A single dose of 20 mg/kg has been shown to result in tumor growth inhibition lasting 20 days (see Example 7 - FIG. 25).
  • This example was conducted as according to the general procedure illustrated in FIG. 24 to evaluate the antitumoral efficacy of repeated dosing of ALD101 (10 mg/kg twice at a 10-day interval) compared to single administration (10 or 20 mg/kg once) in this in vivo castrated model.
  • Antibody drug conjugate ALD101 was diluted in 20 mM histidine, pH 5.5 or in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH5.5.
  • ALD101 solutions were prepared as described in Example 3. Cell lines, cell culture, inoculation and animal experiments were also as described in Example 3.
  • Randomization was performed for antitumor evaluation when the mean tumor volume reached approximately 200 mm 3 (range 104-227 mm 3 ).
  • the mice were randomized according to their tumor volume to ensure that the tumor volume in different groups was similar.
  • the date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
  • Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. Tumor volumes were measured three times per week. When tumors reached a volume of approximately 200 mm 3 (approximately 2-3 weeks after injection), the mice were randomly assigned to a treatment group or vehicle and treated as described in Table 18.
  • Table 18 ADC treatment plan to evaluate antitumor efficacy at a single versus repeated ADC doses in an LNCaP-abl model
  • xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm 3 or after 70 days, and animals were sacrificed. Tumor tissue was then collected from each mouse for formalin- fixed paraffin-embedded (FFPE) blocks for immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry.
  • FFPE formalin- fixed paraffin-embedded
  • Tumor growth curves (presented as mean ⁇ SEM) and mouse body weight (presented as mean ⁇ SD) for each treatment group from treatment start until study termination were measured are presented in FIG. 25 and 26, respectively. Animals presenting signs of tumor ulcerations/necrosis were sacrificed and were excluded from grouped analyses
  • ALD101 showed antitumor effects in vivo in a castrated LNCaP-abl CDX immunodeficient NRG model with a mean tumor volume of 200 mm 3 (range 104-227 mm 3 ) at the time of treatment.
  • Major tumor inhibition was seen in all mice treated with two doses of ALD101 10 mg/kg, with mean tumor growth inhibition (TGI) > 90% for 14 days, as well as in mice treated with a single dose of ALD101 20 mg/kg with mean TGI > 90% for 9 days.
  • TGI tumor growth inhibition
  • This example was performed according to the general procedure outlined in FIG. 27 to demonstrate the antitumoral efficacy of repeated dosing of ALD101 at 10 or 15 mg/kg administered at a 10-day interval compared to a single dose of 20 mg/kg in this in vivo model.
  • Cell lines, cell culture, inoculation and animal experiments were also as described in Example 3.
  • Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. ALD101 solutions were prepared as described in Example 3.
  • mice were castrated 10 days before tumor cell injection.
  • a single preparation of 3xl0 6 LNCaP-abl cells was injected subcutaneously (sc) in the dorsal mouse flank.
  • Xenograft monitoring started 1 week after PC cell injection and was performed three times per week.
  • tumors reached a volume of approximately 300 mm 3 (range -150-500 mm 3 ) approximately 2-3 weeks after injection, the mice were randomized according to their tumor volume and assigned to a treatment group or vehicle and treated as described in Table 19. The date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
  • DO Day 0
  • Table 19 ADC treatment plan to evaluate antitumor efficacy at a single versus repeated ADC doses in an LNCaP-abl CDX model
  • FIGS. 28 and 29 Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 28 and 29, respectively. Data are presented as mean ⁇ SEM or mean ⁇ SD respectively.
  • Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 and prepared according to the general procedure outlined in Example 3.
  • This study was conducted according to the general procedure illustrated in FIG. 42 to evaluate the antitumoral efficacy of repeated dosing of ALD101 at 10 mg/kg administered at 10 days interval compared to a single dose of 10 or 20 mg/kg in a 22Rvl CDX model expressing intermediate and heterogeneous levels of PSMA which differentiates from the high PSMA expressing LNCaP-abl CDX model described above.
  • mice were castrated ten days before tumor cell injection.
  • a single preparation of 2xl0 6 22Rvl cells was injected subcutaneously (sc) in the dorsal mouse flank.
  • Xenograft monitoring started 1 week after PC cell injection and was performed three times per week.
  • tumors reached a volume of approximately 200 mm 3 (range -100-350 mm 3 ) roughly 3 weeks after injection, the mice were randomly assigned to a treatment group or vehicle according to their tumor volume to ensure that the tumor volume in different groups was similar and treated as described in Table 23.
  • the date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
  • Table 23 ADC treatment plan to evaluate antitumor efficacy at a single versus repeated ADC doses in a 22Rvl Model.
  • xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm 3 or after 79 days, and animals were sacrificed.
  • FIGS. 43 and 44 Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 43 and 44, respectively. Data are presented as mean ⁇ SEM and mean ⁇ SD respectively.
  • TGI tumor growth inhibition
  • Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 and prepared according to the general procedure outlined in Example 3.
  • mice were castrated 10 days before tumor cell injection.
  • a single preparation of 2-3 xlO 6 22Rvl cells was injected subcutaneously (sc) in the dorsal mouse flank.
  • Xenograft monitoring started 1 week after PC cell injection and was performed three times per week.
  • tumors reached a volume of approximately 200 mm 3 , roughly 3-4 weeks after injection, the mice were randomly assigned to a treatment group or vehicle according to their tumor volume to ensure that the tumor volume in different groups was similar, and treated as described in Table 24.
  • the date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
  • Table 24 ADC treatment plan to evaluate antitumor efficacy at a single versus repeated
  • FIGS. 46 and 47 Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 46 and 47, respectively. Data are presented as mean ⁇ SEM or mean ⁇ SD respectively.
  • ALD101 LNCaP-abl and 22Rvl prostate cancer (PC) cell lines exposed to ALD101, using fluorescence-activated cell sorting (FACS) and immunofluorescence.
  • FACS fluorescence-activated cell sorting
  • Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5.
  • ADC-Control (ADC-ctrl) was formulated in 20 mM histidine, pH5.5.
  • the ADC-Control was a human IgGl Kappa isotype CTL antibody that did not bind to PSMA conjugated to LD038. Formulations were prepared in the biosafety cabinets.
  • the LNCaP-abl human PC cell line was a gift from Prof. Myles Brown (Dana- Farber Cancer Institute, Boston).
  • the LNCaP-abl cell line was cultured in phenol red-free-RPMI 1640 (cat# 11835063, Life Technologies) supplemented with 10% charcoal- stripped serum (CSS, PAN-Biotech GmbH, Aidenbach, Germany) and 1% penicillin-streptomycin.
  • the 22Rvl cell line was purchased from American Tissue Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium with L-glutamine (cat# 21875-034, Life Technologies) supplemented with 10% fetal bovine serum (FBS-11A, Capricorn Scientific) and 1% penicillin/ streptomycin (cat# 15140-122, Life Technologies).
  • LNCaP-abl and 22Rvl cells were seeded at 1.5xl0 6 cells/well in a 6 well plate and grown until ⁇ 85% confluence.
  • ALD101 or ADC-ctrl were conjugated with fluorochrome- conjugated secondary antibodies by incubating each ADC for 30 minutes at room temperature (RT) with an anti-human IgG Fc fragment specific conjugated with Alexa Fluor® 647-labelled AffiniPure goat anti-human IgG, Fc fragment specific (cat# 109-605-098, Jackson ImmunoResearch) diluted 1 : 100 in 1% BSA in PBS.
  • LNCaP-abl and 22Rvl cells were seeded at 2.5xl0 4 cells/well in a p-slide 8-well ibitreat (#80826, IBIDI GmbH) and grown until ⁇ 85% confluence. Cells were incubated with ALD101 for 1 hour at 4°C to allow for ADC binding while preventing its internalization.
  • cells were either immediately fixed (for TO timepoint) with 4% (w/v) formaldehyde at RT for 20 minutes or incubated for the indicated time points (0.5, 1, 3, or 6 hours) in the incubator at 37°C and then fixed. After fixation, cells were incubated with glycine-based blocking solution (0.1M in 1% of PBS) for 10 min at RT. Cells were washed three times with 3% BSA in lx PBS for 10 min at RT.
  • glycine-based blocking solution 0.1M in 1% of PBS
  • Cells were then permeabilized and stained for 30 min at RT by using permeabilization solution (0.05% saponin, 1% BSA in 1 x PBS) containing 1 : 100 of secondary antibody reagent anti-human IgG Fc fragment specific conjugated with Alexa fluor 647 (cat# 109-605-098, Jackson ImmunoResearch Laboratories) and Alexa Fluor 488 conjugated anti-human LAMPl/CD107a (#IC7985G; Bio-Techne AG) or phalloidin (Alexa Fluor plus 555; #A30106, Thermo Fisher Scientific) before mounting the coverglass. Cells were washed three times with 3% BSA in PBS and once with PBS. Stained slides were mounted with coverglasses using the VECTASHIELD antifade mounting medium containing DAPI (#VC-H-1200, Adipogen AG). Photos were acquired with a Leica Stellaris 5 microscope.
  • FIGS. 56A and 56C, and 56B and 56D Internalization by immunofluorescence and FACS are shown in FIGS. 56A and 56C, and 56B and 56D, respectively. Colocalization with lysosomes by immunofluorescence is shown in FIGS. 57A-57B. Internalization of ALD101 bound to the cell surface was seen as early as 1 hour after treatment (FIG. 56). ALD101 colocalized with lysosomes as soon as 1 hour after binding (FIG. 57). ALD101 internalization was evident in both the LNCaP-abl cell line, which shows high and homogeneous PSMA expression (FIGS. 56A-56B and 57A), and the 22Rvl cell line which has intermediate and heterogenous PSMA expression (FIGS. 56C-56D and FIG. 57B).
  • This study was conducted according to the general procedure illustrated in FIG. 58 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan), total IgG, and total ADC in mouse tumor tissue and plasma after the administration of ALD101 10 mg/kg in an LNCaP-abl CDX castrated immunodeficient mouse model, either NRG (NOD- Raglnull IL2rgnull) or NSG (NOD.Cg-PrkdcSCID I12rgtmlWjl/SzJ) using pooled data from 3 studies.
  • PK pharmacokinetic
  • Total antibody is defined as the totality of anti-PSMA antibodies that are either unconjugated (for example, not conjugated to any payload molecules) or conjugated to at least one payload molecule.
  • Total ADC is defined as anti-PSMA antibodies conjugated to one or several payloads.
  • Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 or in 20 mM histidine, pH 5.5. See Example 3 and Example 10 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions. In each study, 3xl0 6 LNCaP-abl cells were inoculated per mouse, and randomization was performed when mean tumor volume was -400 or 650 mm 3 .
  • Xenograft monitoring started 1 week after injection of LNCaP-abl PC cells and tumor volumes were measured 3 times per week. As soon as tumors reached a volume of 400, 480 or 650 mm 3 (approximately 2-3 weeks after injection), the mice were randomized to ensure comparable tumor volumes in different groups, and treated as described in Table 27. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 27.
  • Table 27 Experimental plan for PK evaluation after single administration of ALD101 10 mg/kg
  • Linker digestion was performed overnight at 37 °C. The reaction was quenched by TFA 20%. Ten microliters of [ 2 Hs]-exatecan (100 ng/mL) were added as an IS. Samples were vortexed and 500 pL methanol and 0.1% formic acid was added to precipitate proteins. Samples were centrifuged at 15000 ref for 10 minutes at 4°C. The supernatants were transferred into clean tubes and evaporated under gentle N2 flux at 37 °C. The residues were reconstituted in 200 pL of a solution of (9: 1 ratio of [water + 0.1% formic acid:methanol + 0.1% formic acid]), then vortexed and centrifuged at 15000 ref for 10 minutes at 4 °C. Supernatants were transferred into an autosampler glass vial and a 5 pL sample was injected into the HPLC- MS/MS system.
  • the calibration curve for total IgG was built with a blank plasma from mice, a zero-blank plasma, 1 sample at the LLOQ, followed by 4 control plasma samples spiked with 5 pL of different ALD101 working solutions to obtain the following final concentrations: 0.5, 1, 5, 10, and 50 pg ALDlOl/mL.
  • QCs were prepared by spiking control plasma samples with 5 pL of ALD101 working solutions to obtain the following final concentration: 6 pg ALDlOl/mL.
  • the calibration curve for total ADC was built with a blank plasma or homogenized tissue sample from mice, a zero-blank plasma or tissue, 1 sample at the LLOQ, followed by 5 control plasma samples spiked with 5 pL of different ALD101 working solutions to obtain the following final concentrations: 0.1, 0.5, 1, 5, 10, and 50 pg ALDlOl/mL.
  • Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 or in 20 mM histidine, pH 5.5. See Example 3 and Example 10 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
  • Table 28 Experimental plan for PK evaluation after single administration of ALD101 at 3,
  • This study was conducted according to the general procedure illustrated in FIG. 63 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma, as well as for total ADC and total IgG in plasma after one to three administrations of ALD101 7.5 mg/kg 10 days apart, in an LNCaP-abl CDX castrated immunodeficient NRG (NOD-Raglnull IL2rgnull) mouse model. Specifically, this example shows that exatecan does not accumulate in plasma following multiple treatment doses of ALD101 7.5 mg/kg administered 10 days apart.
  • PK pharmacokinetic
  • Free exatecan in mice plasma and tumor as well as total ADC and total IgG in plasma were analyzed at 24 hours after the first ALD1010 dose, 9 days after the 2 nd dose, and 24 hours after the 3 rd dose and is presented in FIGS. 64A-64C and 65 respectively.
  • the mean concentration of free exatecan in plasma was 0.55 ng/mL 24 hours after the first dose treatment, and was below the limit of quantification (0.1 ng/mL) 9 days after the second dose of treatment, supporting the absence of systemic free payload accumulation after multiple doses of ALD101 at 7.5 mg/kg.
  • free exatecan concentration in plasma increased to 0.49 ng/ml, a concentration comparable to that reached 24 hours after the first dose.
  • the free exatecan concentration in tumor tissue was approximately 75x higher in tumor tissue compared to plasma 24 hours after the first dose favoring an excellent therapeutic index (FIGS. 64A-64C). Due to the very limited tumor material remaining following the second and third treatments with ALD101, exatecan tumor concentrations cannot be reported.
  • This study was conducted according to the general procedure illustrated in FIG. 66 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma after one to three administrations of ALD101 5 mg/kg 10 days apart, compared to a single administration at 10 mg/kg in an LNCaP-abl CDX castrated immunodeficient NRG or NSG mouse model. Specifically, this example shows that exatecan does not accumulate in plasma following multiple treatment doses of 5 mg/kg ALD101 administered 10 days apart. This example also illustrates the proportionality of free exatecan in plasma and tumor to the administered dose of ALD101 using pooled data from 4 studies.
  • PK pharmacokinetic
  • Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. See Example 10 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
  • Table 31 Experimental plan for PK evaluation after single or repeated administration of
  • Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. See Example 10 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions. See Example 11 for additional information on the source, maintenance and culture of the 22Rvl cell line.
  • Table 33 Experimental plan for PK evaluation after single administration of ALD101 10 mg/kg in a 22Rvl CDX mouse model
  • mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC- MS/MS analysis.
  • Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5.
  • An isotype control ADC (IgGl) that does not bind PSMA was used as a control.
  • the CTRL ADC was formulated in the same buffer. See Example 11 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the 22Rvl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
  • This study was conducted according to the general procedure illustrated in FIG. 79 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma after a single administration of ALD101 10 mg/kg in a patient- derived xenograft mouse model of castration-resistant prostate cancer (C5 model) immunodeficient NRG NOD-Raglnull IL2rgnull.
  • PK pharmacokinetic
  • Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. See Example 11 and Example 17 for additional information on the description of the ADC used (ALD101), ADC administration and manipulation, the source and maintenance of the C5 cell line, animal handling procedures (including protocol ethics committee approval), tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
  • mice were inoculated with 4xl0 6 PDX C5 cells.
  • Cells were injected subcutaneously into the mouse flank using 1 : 1 Matrigel® Matrix (cat# 354234, Coming) with PBS.
  • Xenograft monitoring started 1 week after injection of C5 PC cells and tumor volumes were measured 3 times per week. As soon as tumors reached a volume of 200 mm 3 (roughly 4 weeks after injection), the mice were randomized to ensure comparable tumor volumes in the different groups, and treated as described in Table 36.
  • the date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0.
  • the treatment groups analyzed in this study are presented in Table 36.
  • Table 36 Experimental plan for PK evaluation after single administration of ALD101 10 mg/kg in a PDX C5 mouse model
  • mice Six mice were initially treated but one mouse was excluded due to a problem during ALD101 injection. Thus 5 mice were included in the analysis.

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Abstract

The present disclosure provides anti-PSMA conjugates for use in the treatment of cancer.

Description

ANTLPSMA CONJUGATES AND METHODS OF USING THE SAME
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0001] The contents of the electronic sequence listing (720217 401WO SL. xml; Size: 19,076 bytes; and Date of Creation: November 7, 2024) is herein incorporated by reference in its entirety.
BACKGROUND
[0002] Prostate cancer ranks among the top for the number of new cancer cases in men in the United States. For clinical local diseases, surgery and radiotherapy are usually the options. For locally advanced or metastatic diseases, surgical or chemical androgen deprivation therapy (ADT) is usually the first treatment. Sipuleucel-T cell immunotherapy can be selected for the early stage of castration-resistant prostate cancer (CRPC), and drug therapies such as androgen inhibitors, androgen receptor antagonists, radiotherapy drugs for bone metastasis, and chemotherapy drugs acting on microtubules can be selected for the patients with metastatic castration-resistant prostate cancer (mCRPC) as appropriate. However, each of the therapies can only prolong survival by a few months, so it is necessary to seek effective therapies.
[0003] Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein expressed at high levels in prostate adenocarcinomas and at even higher levels in CRPCs. PSMA is one of the most clinically validated targets for diagnosis and treatment of prostate cancer. In addition to prostate epithelial cells, PSMA can also be expressed by nonprostate tissues, such as the small intestine, proximal renal tubules, and salivary glands, but at levels much lower than in the prostate tissue. PSMA is highly expressed in prostate cancer cells, particularly in metastatic diseases, hormone refractory diseases and high-grade lesions. In addition, PSMA is also highly expressed in endothelial cells of neovasculature of all solid tumors, but not in normal vasculature, so it is a target for the treatment of solid tumors (Clin Cancer Res., 2010; 16(22): 5414-5423). ADT and androgen receptor antagonist therapy can both upregulate the expression of PSMA (J Nucl Med, 2017; 58: 81-84), which provides the basis for targeted therapy in combination with traditional hormone therapy.
[0004] An antibody-drug conjugate (ADC) links a monoclonal antibody or an antibody fragment to a biologically active cytotoxin via a linker compound, making full use of the binding specificity of the antibody to surface antigens of normal cells and tumor cells and the high efficiency of the cytotoxic substance, and also avoiding defects such as poor therapeutic effect of the antibody and serious toxic side effects of the toxic substance. This means that the ADC can kill tumor cells more precisely and has a reduced effect on normal cells compared to conventional chemotherapeutic drugs.
[0005] A variety of ADC drugs have been used in nonclinical or clinical studies, such as Kadcyla, which is an ADC drug composed of Her2 -targeted trastuzumab and drug maytansinoid- 1 (DM1). A number of PSMA-targeted ADC drugs have been tested in clinical therapeutic studies. PSMA-ADC from Progenies Pharmaceuticals which reached phase II clinical testing before being discontinued, while MED 13726 from Medlmmune and MLN2704 from Millennium Pharmaceuticals were discontinued at the end of phase I clinical stage due to poor efficacy. Accordingly, there exists a need for improved ADC drugs targeting PSMA.
BRIEF SUMMARY
[0006] In one aspect, the present disclosure provides a conjugate having the structure of Formula (I):
Ab-[L-E]n
(I) wherein:
Ab is an anti-prostate specific membrane antigen (PSMA) antibody, or an antigenbinding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:4, a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:5; and the VL comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:8;
E is, at each occurrence, an exatecan payload; and
L is a linker covalently attached at a first end to the exatecan payload and further covalently attached at a second end to the Ab via a sulfur-containing residue of the Ab, wherein the linker has, at each occurrence, independently one of the following structures:
Figure imgf000005_0001
Figure imgf000006_0001
wherein:
* is the covalent attachment to the Ab; is the covalent attachment to the exatecan payload; m is about 0 to about 20; and n is about 1 to about 20.
[0007] In some embodiments, the conjugate has the following structure (II):
Figure imgf000006_0002
[0008] In some embodiments, m is about 10 to about 20.
[0009] In some embodiments, the conjugate has the structure of Formula (III):
Figure imgf000007_0001
wherein each L1 independently has one of the following structures:
Figure imgf000007_0002
wherein * is the covalent bond to the Ab and ** is a covalent bond to the carbon marked with an arrow.
Figure imgf000007_0003
, [0013] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody, or de-immunized antibody.
[0014] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2.
[0015] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2, wherein the amino acid sequences of HCDRs 1-3 and LCDRs 1-3 are unchanged.
[0016] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO:2.
[0017] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises: a human immunoglobulin G1 (IgGl) constant region.
[0018] In some embodiments, the anti-PSMA antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.
[0019] In some embodiments, the point of attachment of the linker to the anti-PSMA antibody or antigen binding fragment thereof is a cysteine residue of the anti-PSMA antibody or antigen binding fragment thereof.
[0020] In some embodiments, the point of attachment is a cysteine residue at position 214 of SEQ ID NO: 12, a cysteine residue at position 218 of SEQ ID NO: 11, a cysteine residue at position 224 of SEQ ID NO: 11, and a cysteine residue at position 227 of SEQ ID NO: 11.
[0021] In some embodiments, n is about 4 to about 16.
[0022] In some embodiments, n is about 6 to about 8.
[0023] In some embodiments, n is 6.
[0024] In some embodiments, n is 7.
[0025] In some embodiments, n is 8.
[0026] In some embodiments, n is 9.
[0027] In some embodiments, n is 10. [0028] In another aspect, the present disclosure provides a conjugate formed by reaction of a linker drug with an antibody, or an antigen binding fragment thereof, wherein the linker drug has the following structure:
Figure imgf000009_0001
wherein m is about 0 to about 20; and the antibody, or antigen binding fragment thereof, is an anti-prostate specific membrane antigen (PSMA) antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:4, a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:5; and the VL comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:8, wherein the reacting comprises forming a covalent bond between the sulfur atom of one or more cysteine residues of the antibody, or antigen binding fragment thereof, and one or more linker drugs.
[0029] In some embodiments, m is about 10 to about 20.
[0030] In some embodiments, the linker drug has the following structure:
Figure imgf000010_0001
[0031] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody, or de-immunized antibody.
[0032] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2.
[0033] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2, wherein the amino acid sequences of HCDRsl-3 and LCDRsl-3 are unchanged.
[0034] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO:2.
[0035] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises a human IgGl constant region. [0036] In some embodiments, the anti-PSMA antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.
[0037] In some embodiments, the one or more linker drugs forms a covalent bond with a cysteine residue at position 214 of SEQ ID NO: 12, a cysteine residue at position 218 of SEQ ID NO: 11, a cysteine residue at position 224 of SEQ ID NO: 11, and a cysteine residue at position 227 of SEQ ID NO:11.
[0038] In another aspect, the present disclosure provides a pharmaceutical composition comprising any of the aforementioned conjugates, and a pharmaceutically acceptable excipient.
[0039] In another aspect, the present disclosure provides a method of treating a subject having a PSMA-expressing cancer, comprising administering to a subject in need thereof an effective amount of any of the aforementioned conjugates or the aforementioned pharmaceutical composition.
[0040] In some embodiments, the PSMA-expressing cancer is prostate cancer, salivary gland cancer, thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer.
[0041] In some embodiments, the subject has prostate cancer.
[0042] In some embodiments, the subject has metastatic prostate cancer.
[0043] In some embodiments, the subject has metastatic castrate resistant prostate cancer.
[0044] In some embodiments, the subject has salivary gland cancer.
[0045] In some embodiments, the conjugate is administered systemically.
[0046] In some embodiments, the conjugate is administered intravenously.
[0047] In some embodiments, the conjugate is administered at a dose of about 0.1 mg/kg to about 20 mg/kg.
[0048] In some embodiments, the conjugate is administered at a dose of about 3 mg/kg, at a dose of about 5 mg/kg, at a dose of about 7.5 mg/kg, at a dose of about 10 mg/kg, at a dose of about 15 mg/kg or at a dose of about 20 mg/kg.
[0049] In some embodiments, the conjugate is administered once every 1-3 weeks.
[0050] In some embodiments, the conjugate is administered once every week, once every
2 weeks, or once every 3 weeks.
[0051] In some embodiments, the method further comprises administering an additional therapy. [0052] In some embodiments, the additional therapy comprises surgery, a hormone therapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, a molecularly targeted therapeutic agent, thermotherapy, radiation therapy, or a vaccine.
[0053] In some embodiments, the hormone therapeutic agent is an anti -androgen therapeutic agent.
[0054] In some embodiments, the additional therapeutic agent is bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, proxalutamide, cimetidine, or topilutamide.
[0055] In some embodiments, the additional therapeutic agent is enzalutamide.
[0056] In some other embodiments, the present disclosure provides a method for treating a subject having a PSMA-expressing cancer, comprising administering to a subject in need thereof a predetermined amount of the conjugate of Formula (I), wherein the predetermined amount is administered to the subject in two or more doses within a 30-day period. For example, in some embodiments the predetermined amount is administered to the subject in two doses within the 30-day period, or the predetermined amount is administered to the subject in three doses within the 30-day period. In other embodiments, the predetermined amount is administered to the subject in more than three doses within the 30-day period.
[0057] In some other embodiments, the two or more doses are administered at a 7 to 15- day interval. For example, in some embodiments the two or more doses are administered at a 7- day interval, 8-day interval, 9-day interval or 10-day interval.
[0058] In other embodiments, the two or more doses are administered within a 20-day period. For example, in some embodiments the predetermined amount is administered in three doses within a 20-day period.
[0059] In other embodiments, administration results in an exatecan tumor to plasma ratio in the subject of at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100: 1, at least 110: 1, at least 120: 1, at least 130: 1, at least 140: 1 or at least 150: 1.
[0060] In different embodiments, a period of mean tumor growth inhibition (TGI) in the subject is extended compared to administering the approximately predetermined amount in a single dose. For example, in some embodiments the TGI is extended at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, or at least 400%.
[0061] In other embodiments, the method results in TGI in the subject for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days. [0062] In some embodiments the TGI is 60%, 70%, 80%, 90% or greater.
[0063] In certain embodiments, the method results in complete inhibition of tumor growth for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days.
[0064] In still other embodiments, the subject has a PSMA-expressing cancer with a tumor volume of at least 125 mm3, at least 175 mm3, at least 200 mm3, at least 250 mm3, at least 300 mm3, at least 350 mm3, at least 400 mm3, at least 450 mm3, at least 500 mm3, at least 550 mm3, at least 600 mm3 or at least 650 mm3 prior to administering the two or more doses.
[0065] In some aspects the subject is a human. In other aspects, the PSMA-expressing cancer is prostate cancer, salivary gland cancer, thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer. For example, in some embodiments the subject has prostate cancer, such metastatic prostate cancer or metastatic castrate resistant prostate cancer.
[0066] In other embodiments, the subject has salivary gland cancer.
[0067] In some embodiment the conjugate is administered systemically, and in other embodiments the conjugate is administered intravenously.
[0068] In some embodiments, the method further comprises administering an additional therapy. For example, in some embodiments the additional therapy comprises surgery, a hormone therapeutic agent (e.g., anti-androgen therapeutic agent), a chemotherapeutic agent, an immunotherapeutic agent, a molecularly targeted therapeutic agent, thermotherapy, radiation therapy, or a vaccine. For example, in some embodiments the additional therapeutic agent is bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, proxalutamide, cimetidine, or topilutamide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0069] FIG. 1 is a schematic representation of an embodiment of the antibody drug conjugate molecule of the present disclosure (ALD101). The HUJ591 antibody is conjugated to the linker payload. The chemical structure of the linker payload is shown on the right side. LC refers to the light chain (e.g., SEQ ID NO: 12); HC refers to the heavy chain (e.g., SEQ ID NO: 11).
[0070] FIG. 2 depicts the experimental plan for preparation of single cell suspension of tumor cells from cell-line derived xenografts for fluorescence-activated cell sorting (FACS) analysis. Male mice (NRG mouse model) were castrated and at least 1 week later, were injected subcutaneously (s.c.) with 2xl06 cells of a prostate cancer (PC) cell line (LNCaP-abl). After treatment with vehicle or ALD101 (when tumor volumes reached 125-175 mm3) in antitumor growth experiments tumors were harvested after reaching a volume of 800-1000 mm3 (or a maximum 1500 mm3), for immunohistochemistry (IHC) analysis and digested to create a single cell suspension for flow cytometry analysis.
[0071] FIG. 3 depicts the in vivo experimental plan of treatment in a NOD-Raglnull IL2rgnull (NRG) mouse model. Mice were castrated seven to ten days before tumor cell injection. Cells were injected subcutaneously (s.c.) in the dorsal mouse flank. Two weeks after injection, when the tumor reached approximately 200 mm3, mice were randomized to receive the treatment, a single dose of ALD101, an isotype control nonbinding ADC or vehicle. Mice were monitored for tumor growth and sacrificed when the tumor reached a volume of -800-1000 mm3 (or after 40 days).
[0072] FIG. 4 shows PSMA expression levels on human prostate cancer cell lines as measured by flow cytometry. Fluorescence minus one (FMO) value is represented by a solid black line encompassing a grey histogram. Additional plots are (from left to right): PC3, DU145, 22Rvl, LNCaP-abl and LNCaP. Live cells were discriminated by live/dead dye by fluorescent analysis.
[0073] FIGS. 5A-5C show quantification of PSMA molecules bound per cell measured by flow cytometry using the Quantibrite kit. FIG. 5A: Bead peaks are analyzed using a histogram plot of FL2-H in linear values. FIG. 5B: Linear regression of loglO phycoerythrin (PE) molecules per bead relates to geometric mean fluorescence intensity (GMFI) of each peak. The equation to identify the number of molecules bound per cell is indicated. FIG. 5C: Quantification of PSMA-PE molecules per cell in LNCaP, LNCaP-abl, 22Rvl, PC3, and DU145 cell lines according to the GMFI. PC3 and DU145 are below threshold (defined by the lowest peak).
[0074] FIG. 6 shows immunoblotting analysis of PSMA in prostate cancer cell lines. Representative Western blot analysis of PSMA in LNCaP-abl, LNCaP, 22Rvl, PC3, and DU145 cell lines. GAPDH serves as control.
[0075] FIG. 7 shows comparison of fluorochrome-conjugated anti-human PSMA antibody (BioLegend clone LNL17) vs. unconjugated anti-PSMA antibody (HUJ591). Representative histograms of PSMA expression levels on LNCaP (upper panel), LNCaP-abl (middle panel), and 22Rvl (bottom panel) by flow cytometry. PC cells with different expression levels of PSMA were stained with fluorochrome-conjugated monoclonal antibody (mAb) against PSMA (phycoerythrin-conjugated anti-human PSMA, BioLegend clone LNI-17, left) vs. unconjugated anti-PSMA (HUJ591, right). HUJ591 mAb was incubated with a fluorochrome- conjugated secondary antibody anti-human Fc portion (Alexa Fluor® 647-labeled AffiniPure goat anti-human IgG, Fc fragment specific; Jackson ImmunoResearch. Fluorescence minus one (FMO) value is represented by a grey histogram. Geometric mean fluorescence intensity (GMFI) values are indicated in each plot. Live cells were discriminated by live/dead dye by fluorescent analysis.
[0076] FIG. 8 shows PSMA expression by immunofluorescence with fluorochrome- conjugated mAb against PSMA (phycoerythrin-conjugated anti-human PSMA, BioLegend clone LNI-17) vs. unconjugated anti-PSMA (HUJ591, right). HUJ591 mAb was incubated with a fluorochrome-conjugated secondary antibody anti-human Fc portion (Alexa Fluor® 647-labeled AffiniPure goat anti-human IgG, Fc fragment specific; Jackson ImmunoResearch). Representative immunofluorescence analysis of prostate cancer cell lines with different expression levels of PSMA. LNCaP (upper panel), LNCaP-abl (middle panel), and 22Rvl (bottom panel) were stained with fluorochrome-conjugated anti-human PSMA (left) or unconjugated anti-PSMA (HUJ591, right). Nuclei are stained with DAPI (visible in merged column). Scale bar: 50 pm.
[0077] FIGS. 9A-9D show in vitro cytotoxicity assays in prostate cancer cell lines treated with exatecan. Cell viability assay in LNCaP (FIG. 9A), LNCaP-abl (FIG. 9B), 22Rvl (FIG. 9C), and PC3 (FIG. 9D) cell lines after exatecan treatment for 4 days, using a sulforhodamine B (SRB) assay. IC50 values are indicated. Data are shown as mean ± SD. Each measurement was made in triplicate. Experiment was performed at least n=3.
[0078] FIGS. 10A-10D show ALD101 cytotoxicity in prostate cancer cell lines. Cell viability assay in LNCaP (FIG. 10A), LNCaP-abl (FIG. 10B), 22Rvl (FIG. 10C), and PC3 (FIG. 10D) cell lines after treatment with ALD101 (closed square), nonbinding isotype control ADC (ADC control; open square), or unconjugated HUJ591 mAb (naked mAb; closed circle) for 4 days, using an SRB assay. IC50 values are indicated for LNCaP and LNCaP-abl experiments. Data are shown as mean ± SD. Each measurement was made in triplicate. Experiment was performed at least n=3.
[0079] FIG. 11 shows PSMA expression levels in LNCaP-abl cells (right histogram) before inoculation in mice for cell-line derived xenografts, by flow cytometry. Fluorescence minus one (FMO) is represented by a dark grey histogram on left. Geometric mean fluorescence intensity (GMFI) is indicated in the plot. Live cells were discriminated by live/dead dye by fluorescent analysis. [0080] FIG. 12 shows in vivo antitumor activity of ALD101 in an LNCaP-abl cell-line derived xenograft (CDX) mouse model. Tumor-bearing mice were treated when tumors reached a volume of approximately 125 mm3 with vehicle (black circle), 10 mg/kg nonbinding isotype control ADC (ADC Ctrl; triangle), 3 mg/kg ALD101 (diamond), or 10 mg/kg ALD101 (square). A single dose of each treatment was administered, and mice were monitored for tumor growth 3 times/week. Data are presented as mean ± SEM.
[0081] FIG. 13 shows mouse weight after ALD101 treatment in an LNCaP-abl CDX model. Mouse weight was monitored to evaluate general health status after treatment with vehicle (filled circle), 10 mg/kg nonbinding control ADC (triangle), 3 mg/kg ALD101 (diamond), or 10 mg/kg ALD101 (square). Data are shown as mean ± SD.
[0082] FIGS. 14A-14B show PSMA expression in tumor tissue after ALD101 treatment (upon tumor regrowth). (FIG. 14A) PSMA expression was analyzed by flow cytometry on live cells from tumors collected from CDX mice treated with ALD101 and followed for tumor growth. Bars show the expression levels of PSMA on tumor tissue after treatment with vehicle (left bar), 3 mg/kg ALD101 (middle bar), and 10 mg/kg ALD101 (right bar). Data are shown as mean of geometric mean fluorescence intensity (GMFI) ± SD. Each dot represents a single mouse. (FIG. 14B) Representative immunohistochemical staining on tumor tissue for expression of hematoxylin and eosin (HE; upper panel), Ki-67 (middle panel), and PSMA (lower panel) after treatment with vehicle (left panel), 3 mg/kg ALD101 (middle panel), or 10 mg/kg ALD101 (right panel). Scale bar = 300 pm.
[0083] FIG. 15 shows in vivo antitumor activity of ALD101 in an LNCaP-abl CDX mouse model comparing 10 mg/kg vs 15 mg/kg. Tumor-bearing mice were treated when the tumor reached a volume of approximately 175 mm3 with vehicle (filled circle), 10 mg/kg ALD101 (square), or 15 mg/kg ALD101 (triangle). A single dose of each treatment was administered, and mice were monitored for tumor growth 3 times/week. Data are presented as mean ± SEM.
[0084] FIG. 16 shows mouse weight after ALD101 treatment in an LNCaP-abl CDX model. Mouse weight was monitored to evaluate general health status after treatment with a single dose of vehicle (circle), 10 mg/kg ALD101 (square), or 15 mg/kg ALD101 (light triangle). Data are shown as mean ± SD.
[0085] FIGS. 17A-17D show distribution of exatecan in different body compartments after treatment with 3 and 10 mg/kg ALD101. Exatecan was quantified in biological matrices from LNCaP-abl CDX mice treated with 3 or 10 mg/kg ALD101 (black, 4 mice per group) or nonbinding isotype control ADC (ADC CTRL, white; 3 mice per group), and sacrificed 24 hours later. Exatecan concentration in (FIG. 17A) tumor tissue (ng/g tissue), (FIG. 17B) plasma (ng/mL plasma), (FIG. 17C) liver tissue (ng/g tissue). (FIG. 17D) Ratio of exatecan (per mass tumor versus mass of exatecan/mL plasma).
[0086] FIGS. 18A-18D show distribution of exatecan in different body compartments at 10 and 15 mg/kg ALD101. Exatecan was quantified in different biological matrices from LNCaP-abl CDX mice treated with ALD101 10 or 15 mg/kg or nonbinding isotype control ADC (ADC CTRL), and sacrificed 24 hours later (3 mice per group). (FIG. 18A) tumor tissue, (FIG. 18B) plasma, (FIG. 18C) liver tissue and (FIG. 18D) tumor/plasma ratio. * Student’s T test p- value <0.05.
[0087] FIG. 19 shows an in vivo experimental plan for antitumor efficacy evaluation of a single dose of ALD101 (10 mg/kg) according to tumor size prior to treatment in an LNCaP-abl CDX model per Example 5.
[0088] FIG. 20 shows tumor growth curves for each pre-treatment tumor size group in an LNCaP-abl CDX model treated with a single dose of ALD101 10 mg/kg according to Example 5.
[0089] FIG. 21 shows an in vivo experimental plan for antitumor efficacy evaluation of 3 doses of ALD101 (7.5 mg/kg) in an LNCaP-abl CDX model treated according to Example 6.
[0090] FIG. 22 shows in vivo antitumor activity in mice treated with 3 doses of ALD101 (7.5 mg/kg) or vehicle in an LNCaP-abl CDX model treated according to Example 6. Tumorbearing mice were treated when the tumor reached a volume of approximately 300 mm3.
[0091] FIG. 23 shows mouse weight after 3 doses of ALD101 (7.5 mg/kg) or vehicle treatment in an LNCaP-abl CDX model treated according to Example 6.
[0092] FIG. 24 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated administrations of ALD101 (10, 20 or 10x2 mg/kg) in an LNCaP-abl CDX model treated according to Example 7.
[0093] FIG. 25 shows in vivo antitumor activity of single or repeated doses of ALD101 (10, 20 or 10x2 mg/kg) in an LNCaP-abl CDX mouse model treated according to Example 7. Tumor-bearing mice were treated when the tumor reached a volume of approximately 200 mm3.
[0094] FIG. 26 shows mouse weight after vehicle or single or repeated doses of ALD101 (10, 20 or 10x2 mg/kg) in an LNCaP-abl CDX model treated according to Example 7. [0095] FIG. 27 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (10x2, 15x2 or 20 mg/kg) in an LNCaP-abl CDX model according to Example 8.
[0096] FIG. 28 shows in vivo antitumor activity of single or repeated doses of ALD101 (10x2, 15x2 or 20 mg/kg) in an LNCaP-abl CDX mouse model treated according to Example 8. Tumor-bearing mice were treated when the tumor reached a volume of approximately 300 mm3.
[0097] FIG. 29 shows mouse weight after vehicle or single or repeated doses of ALD101 (10x2, 15x2 or 20 mg/kg) in an LNCaP-abl CDX model treated according to Example 8.
[0098] FIG. 30 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (7.5 mg/kg xl x2 x3 versus 20 mg/kg) in an LNCaP-abl CDX model treated according to Example 9.
[0099] FIG. 31 shows in vivo antitumor activity of single or repeated doses of ALD101 (7.5 mg/kg xl x2 x3 versus 20 mg/kg) in an LNCaP-abl CDX model treated according to Example 9. Tumor-bearing mice were treated when the tumor reached a volume of approximately 200 mm3.
[0100] FIG. 32 shows mouse weight after vehicle or single or repeated doses of ALD101 (7.5 mg/kg xl x2 x3 versus 20 mg/kg) in an LNCaP-abl CDX model according to Example 9.
[0101] FIG. 33 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (5 mg/kg xl x2 x3 versus 15 mg/kg) in an LNCaP-abl CDX model according to Example 10.
[0102] FIG. 34 shows in vivo antitumor activity of single or repeated doses of ALD101 (5 mg/kg xl x2 x3 versus 15 mg/kg) in an LNCaP-abl CDX mouse model treated according to Example 10. Tumor-bearing mice were treated when the tumor reached a volume of approximately 200 mm3.
[0103] FIG. 35 shows mouse weight after vehicle or single or repeated doses of ALD101 (5 mg/kg xl x2 x3 versus 15 mg/kg) in an LNCaP-abl CDX model according to Example 10.
[0104] FIG. 36 shows an in vivo experimental plan for antitumor efficacy evaluation of a single dose of ALD101 (10 or 15 mg/kg) in a 22Rvl CDX model according to Example 11.
[0105] FIG. 37 shows an experimental plan of preparation of tumor tissue single cells for fluorescence-activated cell sorting (FACS) analysis according to Example 11.
[0106] FIG. 38 shows PSMA expression levels in 22Rvl cells prior to inoculation in mice for a cell line-derived xenograft (CDX), as measured by flow cytometry according to Example 11. [0107] FIG. 39 shows in vivo antitumor activity of a single dose of ALD101 (10 or 15 mg/kg) in a 22Rvl CDX mouse model treated according to Example 11. Tumor-bearing mice were treated when the tumor reached a volume of approximately 200 mm3.
[0108] FIG. 40 shows mouse weight after vehicle or a single dose of ALD101 (10 or 15 mg/kg) in a 22Rvl CDX model treated according to Example 11.
[0109] FIGS. 41A-41B show PSMA expression in tumor tissue after a single dose of ALD101 (10 or 15 mg/kg) (upon tumor regrowth) in a 22Rvl CDX model according to Example 11.
[0110] FIG. 42 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (10, 10x2 or 20 mg/kg) in a 22Rvl CDX model according to Example 12.
[0111] FIG. 43 shows in vivo antitumor activity of single or repeated doses of ALD101 (10, 10x2 or 20 mg/kg) in a 22Rvl CDX mouse model treated according to Example 12. Tumorbearing mice were treated when the tumor reached a volume of approximately 200 mm3.
[0112] FIG. 44 shows mouse weight after vehicle or single or repeated doses of ALD101 (10, 10x2 or 20 mg/kg) in a 22Rvl CDX model treated according to Example 12.
[0113] FIG 45 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (7.5 xl, 7.5 x2, 7.5 x3 or 20 mg/kg) in a 22Rvl CDX model according to Example 13.
[0114] FIG. 46 shows in vivo antitumor activity of single or repeated doses of ALD101 (7.5 xl, 7.5 x2, 7.5 x3 or 20 mg/kg) in a 22Rvl CDX mouse model treated according to Example 13. Tumor-bearing mice were treated when the tumor reached a volume of approximately 200 mm3.
[0115] FIG 47 shows mouse weight after vehicle or single or repeated doses of ALD101 (7.5 xl, 7.5 x2, 7.5 x3 or 20 mg/kg) in a 22Rvl CDX model treated according to Example 13.
[0116] FIG 48 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (5 xl, 5 x2, 5 x3 or 15 mg/kg) in a 22Rvl CDX model according to Example 14.
[0117] FIG. 49 shows in vivo antitumor activity of single or repeated doses of ALD101 (5 xl, 5 x2, 5 x3 or 15 mg/kg) in a 22Rvl CDX mouse model treated according to Example 14. Tumor-bearing mice were treated when the tumor reached a volume of approximately 200 mm3.
[0118] FIG. 50 shows mouse weight after vehicle or single or repeated doses of ALD101 (5 xl, 5 x2, 5 x3 or 15 mg/kg) in a 22Rvl CDX model treated according to Example 14. [0119] FIG. 51 shows an in vivo experimental plan for antitumor efficacy evaluation of single or repeated doses of ALD101 (10, 10x2 or 20 mg/kg) in a PDX C5 model according to Example 15.
[0120] FIG 52 shows PSMA expression levels in C5 cells prior to inoculation in mice for a patient-derived xenograft (PDX), as measured by flow cytometry.
[0121] FIG. 53 shows in vivo antitumor activity of single or repeated doses of ALD101 (10, 10x2 or 20 mg/kg) in a PDX C5 mouse model treated according to Example 15. Tumorbearing mice were treated when the tumor reached a volume of approximately 200 mm3.
[0122] FIG. 54 shows mouse weight after vehicle or single or repeated doses of ALD101 (10, 10x2 or 20 mg/kg) in a PDX C5 model treated according to Example 15.
[0123] FIGS. 55A-55B show PSMA expression in tumor tissue after single or repeated doses of ALD101 (10, 10x2 or 20 mg/kg) (upon tumor regrowth) in a PDX C5 model treated according to Example 15.
[0124] FIGS. 56A-56D show the internalization of ALD101 in LNCaP-abl and 22Rvl cells according to Example 16. ALD101 is localized on the membrane (see, for example arrows in FIG. 56A for ADC, Ih) or in the cytoplasm (see, for example arrows in FIG. 56A for ADC, 6h). Scale bar: 10 pm. ALD101 internalization is quantified by FACS 1 and 3 hours after binding (FIG 56B and D).
[0125] FIGS. 57A-57B show internalization and colocalization of ALD101 in lysosomes in LNCaP-abl and 22Rvl cells according to Example 16. Scale bar: 20 pm (upper panel) or 10 pm for magnification (ZOOM).
[0126] FIG. 58 shows an in vivo experimental plan for PK evaluation of a single dose of ALD101 10 mg/kg in the LNCaP-abl CDX model according to Example 17.
[0127] FIGS. 59A-59C show free exatecan in plasma or tumor and tumor/plasma ratio after a single dose of ALD101 10 mg/kg in an LNCaP-abl CDX model according to Example 17.
[0128] FIG. 60 shows total ADC and total antibody in plasma after a single dose of ALD101 of 10 mg/kg in an LNCaP-abl CDX model according to Example 17.
[0129] FIG. 61 shows an in vivo experimental plan for PK evaluation of a single dose of ALD101 (from 3 to 20 mg/kg) in an LNCaP-abl CDX model according to Example 18.
[0130] FIGS. 62A-62C show free exatecan in plasma or tumor and tumor/plasma ratio 24hours after a single dose of ALD101 (from 3 to 20 mg/kg) is administered in an LNCaP-abl CDX model according to Example 18. [0131] FIG. 63 shows an in vivo experimental plan for PK evaluation of single or repeated doses of ALD101 (7.5 mg/kg) in an LNCaP-abl CDX model according to Example 19.
[0132] FIGS. 64A-64C show free exatecan in plasma or tumor after repeated doses of ALD101 7.5 mg/kg in an LNCaP-abl CDX model according to Example 19.
[0133] FIG. 65 shows total ADC and total IgG in plasma after 1 to 3 doses of ALD101 7.5 mg/kg in an LNCaP-abl model according to Example 19.
[0134] FIG. 66 shows an in vivo experimental plan for PK evaluation of single or repeated doses of ALD101 (5 mg/kg xl x2 x3) in an LNCaP-abl CDX model according to Example 20.
[0135] FIGS. 67A-67B show free exatecan in plasma or tumor and tumor/plasma ratio after 1 to 3 doses of ALD101 5 mg/kg in an LNCaP-abl CDX model according to Example 20.
[0136] FIGS. 68A-68C show free exatecan in plasma or tumor and tumor/plasma ratio after 1 to 3 doses of ALD101 5 mg/kg in an LNCaP-abl CDX model (superimposed values for once, twice and thrice 5 mg/kg treatments) according to Example 20.
[0137] FIGS.69A-69C show free exatecan in plasma or tumor and tumor/plasma ratio after ALD101 (single dose 5 or 10 mg/kg) in an LNCaP-abl CDX model according to Example 20.
[0138] FIG. 70 shows an in vivo experimental plan for PK evaluation of ALD101 (single dose 10 mg/kg) in a 22Rvl CDX model according to Example 21.
[0139] FIGS. 71A-71C show free exatecan in plasma or tumor and tumor/plasma ratio 24 hours after ALD101 single dose 10 mg/kg in a 22Rvl CDX model according to Example 21.
[0140] FIG. 72 shows an in vivo experimental plan for PK evaluation of ALD101 (single dose 10 mg/kg) in a 22Rvl CDX model according to Example 22.
[0141] FIGS. 73A-73C show free exatecan in plasma or tumor after ALD101 single dose 10 mg/kg in a 22Rvl CDX model according to Example 22.
[0142] FIG. 74 shows total ADC and total IgG in plasma after a single administration of ALD101 at 10 mg/kg in 22Rvl CDX mice according to Example 22.
[0143] FIG. 75 shows an in vivo experimental plan for PK evaluation of ALD101 or non-binding CTRL ADC (single dose 5 or 7.5 mg/kg) in a 22Rvl CDX model according to Example 23.
[0144] FIGS. 76A-76C show free exatecan in plasma or tumor after ALD101 or nonbinding CTRL ADC single dose 5 or 7.5 mg/kg in a 22Rvl CDX model according to Example 23. [0145] FIG. 77 shows an in vivo experimental plan for PK evaluation of a single dose of ALD101 (from 3 to 20 mg/kg) in two CDX models (LNCaP-abl and 22Rvl) according to Example 24.
[0146] FIGS. 78A-78C show free exatecan in plasma or tumor and tumor/plasma ratio 24hours after a single dose of ALD101 (from 3 to 20 mg/kg) is administered in an LNCaP-abl or 22Rvl CDX model according to Example 24. The dose at 3 and 20 mg/kg were not performed in the 22Rvl CDX model.
[0147] FIG. 79 shows an in vivo experimental plan for PK evaluation of a single dose of ALD101 10 mg/kg in a PDX C5 model according to Example 25.
[0148] FIGS. 80A-80C show free exatecan in plasma or tumor and tumor/plasma ratio 24hours after a single dose of ALD101 10 mg/mL is administered in a PDX C5 model according to Example 25.
DETAILED DESCRIPTION
Definitions
[0149] Prior to setting forth this disclosure in more detail, it may be helpful to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.
[0150] As used in the specification and claims, the singular form “a,” “an,” and “the” includes plural references unless the context clearly dictates otherwise. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components.
[0151] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include” and “comprise” are used synonymously.
[0152] The phrase “at least one of’ when followed by a list of items or elements refers to an open-ended set of one or more of the elements in the list, which may, but does not necessarily, include more than one of the elements.
[0153] The term “about” as used herein in the context of a number refers to a range centered on that number and spanning 15% less than that number and 15% more than that number. The term “about” used in the context of a range refers to an extended range spanning 15% less than that the lowest number listed in the range and 15% more than the greatest number listed in the range. [0154] Throughout this disclosure, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range of the disclosure relating to any physical feature, such as polymer subunits, size, or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. Throughout the disclosure, numerical ranges are inclusive of their recited endpoints, unless specifically stated otherwise.
[0155] Throughout this disclosure, the abbreviations for the natural L-enantiomeric amino acids are conventional and can be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); and valine (V, Vai).
[0156] As used herein, a "variant" protein or polypeptide comprises one or more non-natural amino acids, one or more amino acid substitutions, one or more amino acid insertions, one or more amino acid deletions, or any combination thereof, which may occur at one or more sites relative to a reference polypeptide of this disclosure, and wherein the variant protein or polypeptide has substantially similar activity (e.g., enzymatic function, immunogenicity) relative to a reference polypeptide. A variant protein or polypeptide of this disclosure may have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence for a reference polypeptide of this disclosure as determined by sequence alignment programs and parameters disclosed herein. A variant polypeptide can result from, for example, a genetic polymorphism or by human manipulation. Conservative substitutions of amino acids are well known and may occur naturally or may be engineered when a protein is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY, 2001). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to produce polynucleotides having altered codons that provide the desired substitution, deletion, or insertion. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis or oligonucleotide-directed mutagenesis, may be used to prepare polypeptide variants (see, e.g., Sambrook et al., supra). [0157] As used herein, a protein domain (e.g., a binding domain, a hinge domain, a dimerization or heterodimerization domain, an Fc region constant domain portion) or a protein (which may have one or more domains) “consists essentially of’ a particular amino acid sequence when the amino acid sequence of a protein domain or protein includes extensions, deletions, mutations, or a combination thereof e.g., amino acids at the amino- or carboxyterminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the domain or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, or 5%) the activity of the domain(s) or protein (e.g., the immunological activity of the Fc region constant domain portion or the target binding affinity of a binding protein).
[0158] In some embodiments, a “binding domain” or a “binding region” refers to a protein, polypeptide, oligopeptide, or peptide that is capable of specifically binding to a target (e.g., PSMA). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding domains of this disclosure include, for example, a Fab', F(ab')2, Fab, Fv, rlgG, scFv (single-chain variable fragment), hcAbs (heavy chain antibodies), a single domain antibody, VHH (or nanobody), VNAR (variable new antigen receptor), sdAbs, nanobody, receptor ectodomains or ligand-binding portions thereof, or ligands. A "Fab" (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond. A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, including Western blot, ELISA, and Biacore® analysis. Particularly preferred binding domains comprise immunoglobulin light and heavy chain variable domains (e.g., scFv, Fab) and are herein referred to as "immunoglobulin binding domains" or "immunoglobulin binding proteins." Immunoglobulin binding domains can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.
[0159] As used herein, a "fusion protein" refers to a single chain polypeptide having a binding domain specific for a target (e.g., an antigen binding domain) and at least one other distinct domain, wherein the domains are not naturally found together in a protein. A nucleic acid molecule encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be made synthetically. A fusion protein may further contain other components (e.g., covalently bound), such as a tag, label, or bioactive molecule. In some embodiments, a fusion protein comprises a target binding domain and an Fc region. In some such embodiments, the Fc region is the Fc region of IgGl, such as human IgGl. In other embodiments, the fusion protein binds to two or more targets. In some embodiments, the binding domain binds specifically to multiple targets. In some embodiments, there are multiple distinct binding domains of the fusion protein that bind specifically to two or more targets.
[0160] Throughout this disclosure, the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive toward, an antigen. In certain embodiments, an antibody is an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as an antigen-binding portion of an intact antibody that has or retains the capacity to bind a target molecule. An antibody or an antigen binding fragment thereof of this disclosure can include, for example, polyclonal, monoclonal, and genetically engineered antibodies. A monoclonal antibody or antigen-binding portion thereof of this disclosure can be, for example, non-human (e.g., murine, rabbit), chimeric, humanized, or human. In certain embodiments, an antibody of this disclosure is a heteroconjugate, bispecific, multi-specific, diabody, triabody, or tetrabody. Immunoglobulin structure and function are reviewed, for example, in Greenfield, Ed., Antibodies: A Laboratory Manual, Chapters 2 and 3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 2014).
[0161] As used throughout the disclosure, an “antigen-binding domain” or “antigenbinding fragment” refers to a region of a molecule that specifically binds to an antigen. An antigen binding domain can be an antigen-binding portion of an antibody or an antibody fragment. An antigen-binding fragment can include, for example, a Fab', F(ab')2, Fab, Fv, rlgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, or nanobody.
[0162] For example, the terms “VL” and “VH” refer to the variable region from an antibody light and heavy chain, respectively. The variable regions are made up of discrete, well- defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs). The term “CL” refers to an “immunoglobulin light chain constant region” or a “light chain constant region,” i.e., a constant region from an antibody light chain. The term “CH” refers to an “immunoglobulin heavy chain constant region” or a “heavy chain constant region,” which is further divisible, depending on the antibody isotype into CHI, CH2, and CH3 (IgA, IgD, IgG), or CHI, CH2, CH3, and CH4 domains (IgE, IgM). [0163] As used throughout the disclosure, an “Fc domain” refers to a domain from an Fc portion of an antibody that can specifically bind to an Fc receptor, such as an Fey receptor or an
FcRn receptor.
[0164] A binding domain and a fusion protein thereof “specifically binds” a target if it binds the target with an affinity or Ka (z.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 105 M’1, while not significantly binding other components present in a test sample. Binding domains (or fusion proteins thereof) may be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof). “High affinity” binding domains refer to those binding domains with a Ka of at least 108 M’1, at least 109 M’1, at least IO10 M’1, at least 1011 M’1, at least 1012 M’1, or at least 1013 M’1, preferably at least 108 M'1 or at least 109 M'1. “Low affinity” binding domains refer to those binding domains with a Ka of up to 108 M’1, up to 107 M’1, up to 106 M’1, up to 105 M'1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10'5 M to 10’ 13 M). Affinities of binding domain polypeptides and fusion proteins according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51 :660, 1949; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
[0165] As used throughout the disclosure, “PSMA,” also known as “prostate-specific membrane antigen,” “Glutamate carboxypeptidase II” (GCPII), “N-acetyl-L-aspartyl-L- glutamate peptidase I” (NAALADase I), “NAAG peptidase,” or “folate hydrolase 1” (FOLH1), refers to a transmembrane type II glycoprotein that catalyzes the hydrolysis of N- acetylaspartylglutamate (NAAG) to glutamate and N-acetylaspartate (NAA). PSMA is mainly expressed in the prostate epithelium, proximal tubules of the kidney, the jejunal brush border of the small intestine and ganglia of the nervous system. Higher levels of PSMA expression have been detected in prostate cancer cells. PSMA expression has also been observed in salivary glands cancer, thyroid cancer, hepatocellular carcinoma, tumor associated neovasculature of renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer. PSMA includes mammalian PSMA proteins, e.g., mouse, rat, rabbit, guinea pig, pig, sheep, dog, non-human primate, and human. In some embodiments, PSMA refers to an alternatively spliced variant. In some embodiments, PSMA is a human PSMA having the amino acid sequence set forth in UniProt Reference Q04609-1 (SEQ ID NO:13). [0166] "Derivative," as used herein, refers to a chemically or biologically modified version of a compound that is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. Generally, a "derivative" differs from an "analogue" in that a parent compound may be the starting material to generate a "derivative," whereas the parent compound may not necessarily be used as the starting material to generate an "analogue." An analogue may have different chemical or physical properties of the parent compound. For example, a derivative may be more hydrophilic or it may have altered reactivity (e.g., a CDR having an amino acid change that alters its affinity for a target) as compared to the parent compound.
[0167] As used herein, “identical” or “identity” refer to the similarity between a DNA, RNA, nucleotide, amino acid, or protein sequence to another DNA, RNA, nucleotide, amino acid, or protein sequence. Identity can be expressed in terms of a percentage of sequence identity of a first sequence to a second sequence. Percent (%) sequence identity with respect to a reference DNA sequence can be the percentage of DNA nucleotides in a candidate sequence that are identical with the DNA nucleotides in the reference DNA sequence after aligning the sequences. Percent (%) sequence identity with respect to a reference amino acid sequence can be the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid 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. For example, the percent sequence identity values for sequences provided herein can be generated using the NCBI BLAST 2.0 software as defined by Altschul et al., “Gapped BLAST and PSLBLAST: a new generation of protein database search programs,” Nucleic Acids Res. 1997, 25, 3389-3402, with the parameters set to default values.
Antibody-Drug Conjugates
[0168] In one embodiment of the present invention is provided a conjugate having the structure of Formula (I):
Ab-[L-E]n
(I). wherein
Ab is an anti-PSMA antibody or antigen binding fragment thereof;
L is a linker as described herein; E is an exatecan payload as described herein; and n is 1-20.
Anti-PSMA Antibodies and Antigen-Binding Fragments Thereof
[0169] Provided herein are anti-PSMA antibodies (also referred to as PSMA binding antibodies) and antigen-binding fragments thereof that specifically bind to PSMA for use in conjugates of anti-PSMA antibodies or antigen binding fragments thereof and cytotoxic agents, such as exatecan, also referred to herein as anti-PSMA conjugates. In some embodiments, the anti-PSMA conjugates of the present disclosure reduce the number of PSMA+ cancer cells in a subject.
[0170] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL),
(a) wherein the VH region comprises a heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO:4, and a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 5; and
(b) wherein the VL region comprises a light chain complementary determining region 1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO:6, a light chain complementary determining region 2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 8, wherein the antibody or antigen-binding fragment thereof specifically binds to PSMA.
[0171] In some embodiments, the anti-PSMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2.
[0172] In some embodiments, the anti-PSMA antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2, provided that the amino acid sequences of the HCDRs 1-3 and LCDRs 1-3 are unchanged.
[0173] In some embodiments, the anti-PSMA antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:2.
[0174] In some embodiments, an anti-PSMA antibody or antigen binding fragment thereof may be identified by its heavy and/or light chain CDRs using any one of the following methods: Kabat, Chothia, AbM, Contact, IMGT, and/or Aho. In some embodiments, an anti- PSMA antibody or antigen-binding fragment thereof may be identified by CDRs, variable regions, and heavy and light chains provided in Table 1. The CDRs shown in Table 1 are as defined by Kabat numbering.
Table 1: Anti-PSMA antibody sequences
Figure imgf000029_0001
Figure imgf000030_0001
[0175] In some embodiments of the disclosure, an antibody or antigen-binding fragment thereof comprises two light chain polypeptides (light chains) and two heavy chain polypeptides (heavy chains), held together covalently by disulfide linkages.
[0176] In some embodiments, the heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region. In some embodiments, the heavy chain constant region typically comprises three domains, CHI, CH2, and CH3. Nonlimiting exemplary heavy chain constant regions include human IgGl, human IgG2, human IgG3, and human IgG4 constant regions. In some embodiments, an antibody of the disclosure comprises an IgGl constant region. An exemplary heavy chain constant region includes human IgGl heavy chain constant region (SEQ ID NO: 15).
[0177] In some embodiments the light chain comprises a light chain variable region (VL) and a light chain constant region. Nonlimiting exemplary light chain constant regions include kappa and lambda constant regions. A nonlimiting exemplary human kappa constant region is shown in SEQ ID NO: 14.
[0178] The constant domains provide the general framework of the antibody and may not be involved directly in binding the antibody to an antigen, but can be involved in various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC), ADCP (antibody-dependent cellular phagocytosis), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors (e.g., CD16, CD32, FcRn), greater in vivo half-life relative to a polypeptide lacking an Fc region, protein A binding, and perhaps even placental transfer (see Capon et al., Nature 337:525, 1989). As used throughout the disclosure, "an Fc region constant domain portion" or "Fc region portion" refers to the heavy chain constant region segment of the Fc fragment (the "fragment crystallizable" region or Fc region) from an antibody, which can in include one or more constant domains, such as CH2, CH3, CH4, or any combination thereof. In some embodiments, an Fc region portion includes the CH2 and CH3 domains of an IgG, IgA, or IgD antibody and any combination thereof, or the CH3 and CH4 domains of an IgM or IgE antibody and any combination thereof.
[0179] An Fc region or domain may interact with different types of FcRs. The different types of FcRs may include, for example, FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcaRI, FcpR, FcaRI, FcsRII, and FcRn. FcRs may be located on the membrane of certain immune cells including, for example, B lymphocytes, natural killer cells, macrophages, neutrophils, follicular dendritic cells, eosinophils, basophils, platelets, and mast cells. Once the FcR is engaged by the Fc domain, the FcR may initiate functions including, for example, clearance of an antigenantibody complex via receptor-mediated endocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), trogocytosis, trogoptosis, and ligand-triggered transmission of signals across the plasma membrane that can result in alterations in secretion, exocytosis, and cellular metabolism. FcRs may deliver signals when FcRs are aggregated by antibodies and multivalent antigens at the cell surface. The aggregation of FcRs with immunoreceptor tyrosine-based activation motifs (IT AMs) may sequentially activate SRC family tyrosine kinases and SYK family tyrosine kinases. ITAM comprises a twice-repeated YxxL sequence flanking seven variable residues. The SRC and SYK kinases may connect the transduced signals with common activation pathways.
[0180] In some embodiments, an Fc region or domain can exhibit reduced binding affinity to one or more Fc receptors. In some embodiments, an Fc region or domain can exhibit reduced binding affinity to one or more Fey receptors. In some embodiments, an Fc region or domain can exhibit reduced binding affinity to FcRn receptors. In some embodiments, an Fc region or domain can exhibit reduced binding affinity to Fey and FcRn receptors. In some embodiments, an Fc domain is an Fc null domain or region. As used throughout the disclosure, an “Fc null” refers to a domain that exhibits weak to no binding to any of the Fey receptors. In some embodiments, an Fc null domain or region exhibits a reduction in binding affinity (e.g., increase in Kd) to Fey receptors of at least about 1000-fold.
[0181] The Fc region or domain may have one or more, two or more, three or more, or four or more, or up to five amino acid substitutions that decrease binding of the Fc region or domain to an Fc receptor. In some embodiments, an Fc region or domain exhibits decreased binding to FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD 16a), FcyRIIIB (CD 16b), or any combination thereof. In order to decrease binding affinity of an Fc region or domain to an Fc receptor, an Fc region or domain may comprise one or more amino acid substitutions that has the effect of reducing the affinity of the Fc domain or region to an Fc receptor. In some embodiments, the Fc region or domain is an IgGl and the one or more substitutions in the Fc region or domain comprise any one or more of IgGl heavy chain mutations corresponding to E233P, L234V, L234A, L235A, L235E, AG236, G237A, E318A, K320A, K322A, A327G, A330S, or P331 S according to the EU index of Kabat numbering.
[0182] In some embodiments, the Fc region or domain can comprise a sequence of the IgGl isoform that has been modified from the wild-type IgGl sequence. A modification can comprise a substitution at more than one amino acid residue, such as at 5 different amino acid residues including L235V/F243L/R292P/Y300L/P396L (IgGIVLPLL) according to the EU index of Kabat numbering. A modification can comprise a substitution at more than one amino acid residues, such as at two different amino acid residues including S239D/I332E (IgGIDE) according to the EU index of Kabat numbering. A modification can comprise a substitution at more than one amino acid residue, such as at three different amino acid residues including S298A/E333A/K334A (IgGl AAA) according to the EU index of Kabat numbering. A non- limiting exemplary human IgGl heavy chain constant regions is shown in SEQ ID NO: 15. In some embodiments, an antibody of the disclosure comprises a mouse IgG2a heavy chain constant region. In some embodiments, an antibody of the disclosure comprises a rat IgG2b heavy chain constant region.
[0183] An antibody or Fc domain may be modified to acquire or improve at least one constant region-mediated biological effector function relative to an unmodified antibody or Fc domain, e.g., to enhance FcyR interactions. In some embodiments, a modification can increase CD32b binding (and support transdelivery in a PBMC assay) comprises a substitution at S267L and E329F (IgGILF, also known as SELF double mutant) according to the EU index of Kabat numbering. For example, an antibody with a constant region that binds to FcyRIIA, FcyRIIB and/or FcyRIIIA with greater affinity than the corresponding wild type constant region may be produced according to the methods described of the disclosure. An Fc domain that binds to FcyRIIA, FcyRIIB and/or FcyRIIIA with greater affinity than the corresponding wild type Fc domain may be produced according to the methods of the disclosure.
[0184] In some embodiments, an Fc region or domain found in an anti-PSMA antibody of the disclosure is capable of mediating one or more of these effector functions, or lacks one or more or all of these activities or have one or more of the effector activities increased by way of, for example, one or more mutations as compared to the unmodified Fc region or domain.
[0185] In some embodiments, the antigen-binding regions of the antibody variable regions (VH and VL) comprise HCDRsl-3 disposed within a heavy chain framework region and LCDRsl-3 disposed within a light chain framework region.
[0186] In some embodiments of the disclosure, including any of the aforementioned embodiments, the VH, for example the VH comprising SEQ ID NO: 1, may be joined to a heavy chain constant region, such as a human IgGl constant region of SEQ ID NO: 15.
[0187] In some embodiments of the disclosure, including any of the aforementioned embodiments, the VL, for example the VL comprising SEQ ID NO:2, may be joined to a light chain constant region, such as a human kappa constant region of SEQ ID NO: 14.
[0188] In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12, provided that the amino acid sequences of the HCDRs and LCDRs are unchanged. In one embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO: 11 without the C-terminal lysine residue, and the light chain comprises the amino acid sequence of SEQ ID NO: 12.
[0189] In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and a light chain comprising the amino acid sequence of SEQ ID NO: 12.
[0190] In some embodiments of the disclosure, an anti-PSMA antibody or antigen binding fragment thereof can be a murine, chimeric, humanized, or de-immunized antibody. Chimeric and humanized forms of non-human (e.g., murine) antibodies can be intact (full length) chimeric immunoglobulins, immunoglobulin chains or antigen binding fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subdomains of antibodies), which can contain sequences derived from non-human immunoglobulin. In some embodiments, a chimeric antibody contains non-human VH and VL regions and human constant regions. In some embodiments, the humanized antibody or antigen binding fragment thereof can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), an Fc domain. In some embodiments, including those in which a humanized antibody can also comprise at least a portion of an Fc domain, the Fc domain comprises a human immunoglobulin sequence. A de-immunized antibody refers to an antibody that has been modified to reduce immunogenicity of the antibody. Examples of deimmunization strategies include site-directed mutagenesis to remove B-cell epitopes or T-cell epitopes. Non-specific shielding approaches for deimmunization include PEGylation, fusion to polypeptides (e.g, XTEN or PAS), reductive methylation, glycosylation, and polysialylation.
[0191] In some embodiments of the disclosure, an anti-PSMA antibody or antigen binding fragment thereof of the disclosure comprises a human antibody. As used throughout the disclosure, “human antibodies” can include antibodies having, for example, the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that typically do not express endogenous immunoglobulins. Human antibodies can be produced using transgenic mice incapable of expressing functional endogenous immunoglobulins, but capable of expressing human immunoglobulin genes. Completely human antibodies that recognize a selected epitope can be generated using guided selection. In this approach, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
[0192] In some embodiments of the disclosure, an anti-PSMA antibody or antigen binding fragment thereof comprises a derivatized or otherwise modified sequence. In some embodiments of the disclosure, an anti-PSMA antibody is a derivatized antibody. In some embodiments, an anti- PSMA antibody comprises a derivatized antibody or an antigen binding fragment thereof. In some embodiments of the disclosure, an anti-PSMA antibody is a modified antibody. In some embodiments, an anti- PSMA antibody comprises a modified antibody or an antigen binding fragment thereof. For example, derivatized antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or the like.
Linkers
[0193] In one embodiment is proved a linker covalently attached at a first end to the exatecan payload and further covalently attached at a second end to the Ab via a sulfur- containing residue of the Ab, wherein the linker has, at each occurrence, independently one of the following structures:
Figure imgf000035_0001
Figure imgf000036_0001
wherein:
* is the covalent attachment to the Ab; is the covalent attachment to the exatecan payload; and m is about 0 to about 20.
[0194] In one embodiment, m is about 10 to about 20. In a further embodiment, m is 11. In another embodiment, m is 13. In another embodiment, m is 15. In another embodiment, m is 17. In another embodiment, m is 19. Payload
[0195] In one embodiment, the cytotoxic agent used in the present invention is exatecan, a camptothecin derivative, ((15,95)-l-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-l,2,3,9,12,15- hexahydro- 1 OH, 13Z/-benzo[t/e]pyrano[3 ',4' : 6,7]indolizino[ 1 ,2-Z>]quinoline- 10,13 -di one), having the structure of Compound 101 :
Figure imgf000037_0001
Compound 101.
[0196] In one embodiment, Compound 101 is covalently attached to the linker through the amino group at position 1.
[0197] In another embodiment, the conjugate has the structure of Formula (II):
Figure imgf000037_0002
wherein
Ab is an anti-PSMA antibody;
L is a linker as described herein; and n is 1-20.
[0198] In one embodiment is provided a conjugate having the structure of Formula (III):
Figure imgf000038_0001
wherein each L1 independently has one of the following structures:
Figure imgf000038_0002
wherein * is the covalent bond to the Ab and ** is a covalent bond to the carbon marked with an arrow.
Figure imgf000038_0003
Linker-Drug Conjugation
[0199] In one embodiment, a conjugate is formed by reaction of a linker drug with an antibody, or an antigen binding fragment thereof, wherein the linker drug has the following structure:
Figure imgf000039_0001
wherein m is about 0 to about 20; and the antibody, or antigen binding fragment thereof, is an anti-prostate specific membrane antigen (PSMA) antibody, or an antigen-binding fragment thereof.
[0200] In one embodiment, the reacting comprises forming a covalent bond between the sulfur atom of one or more cysteine residues of the antibody, or antigen binding fragment thereof, and one or more linker drugs.
[0201] In one embodiment, the one or more linker drugs forms a covalent bond with a cysteine residue at one or more of the following positions: position 214 of the antibody light chain, position 218 of the antibody heavy chain, position 224 of the antibody heavy chain, position 227 of the antibody heavy chain, or any combination thereof. The positions of the cysteine residues in the antibody light chain and antibody heavy chain may be made in reference to SEQ ID NO: 12 and SEQ ID NO: 11, respectively. In one embodiment, the one or more linker drugs forms a covalent bond with a cysteine residue at position 214 of the antibody light chain, position 218 of the antibody heavy chain, position 224 of the antibody heavy chain, and position 227 of the antibody heavy chain. [0202] In one embodiment, m is about 10 to about 20. In a further embodiment, m is 11.
In another embodiment, m is 13. In another embodiment, m is 15. In another embodiment, m is
17. In another embodiment, m is 19.
[0203] In one embodiment, a conjugate is formed by reaction of a linker drug with an antibody, or an antigen binding fragment thereof, wherein the linker drug has the following structure:
Figure imgf000040_0001
wherein the antibody, or antigen binding fragment thereof, is an anti-prostate specific membrane antigen (PSMA) antibody, or an antigen-binding fragment thereof.
[0204] In one embodiment, the reacting comprises forming a covalent bond between the sulfur atom of one or more cysteine residues of the antibody, or antigen binding fragment thereof, and one or more linker drugs.
[0205] In one embodiment, the one or more linker drugs forms a covalent bond with a cysteine residue at one or more of the following positions: position 214 of the antibody light chain, position 218 of the antibody heavy chain, position 224 of the antibody heavy chain, position 227 of the antibody heavy chain, or any combination thereof. In one embodiment, the one or more linker drugs forms a covalent bond with a cysteine residue at one or more of the following positions: position 214 of the antibody light chain, position 218 of the antibody heavy chain, position 224 of the antibody heavy chain, position 227 of the antibody heavy chain, or any combination thereof. The positions of the cysteine residues in the antibody light chain and antibody heavy chain may be made in reference to SEQ ID NO: 12 and SEQ ID NO: 11, respectively. In one embodiment, the one or more linker drugs forms a covalent bond with a cysteine residue at position 214 of the antibody light chain, position 218 of the antibody heavy chain, position 224 of the antibody heavy chain, and position 227 of the antibody heavy chain.
[0206] In one embodiment, the antibody, or antigen binding fragment thereof, is an antiprostate specific membrane antigen (PSMA) antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:4, a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:5; and the VL comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:8.
[0207] In certain embodiments, when the conjugate is administered to a patient having an intermediate or heterogeneous PSMA -expressing cancer, the conjugate provides a tumor to plasma ratio of free exatecan that is within 30% of the tumor to plasma ratio of free exatecan when the conjugate is administered to a patient having a high PSMA -expressing cancer, preferably wherein the tumor to plasma ratio of free exatecan is within 25%, 20%, 15%, 10% or 5%. In some of these embodiments, the tumor plasma ratio of free exatecan is higher when the conjugate is administered to a patient having an intermediate or heterogeneous PSMA - expressing cancer compared to the tumor to plasma ratio of free exatecan when the conjugate is administered to a patient having a high PSMA -expressing cancer.
Pharmaceutical Formulations
[0208] Anti-PSMA conjugates and compositions thereof of this disclosure are useful as, or may be used in, pharmaceutical compositions for administration to a subject in need thereof. In some embodiments, pharmaceutical compositions comprise a conjugate of this disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients. In further embodiments, a pharmaceutical composition comprises at least one of the conjugates of this disclosure and further comprises one or more of a buffer, buffers, carbohydrates, and/or preservatives, as appropriate.
[0209] Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries. Formulations may be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a conjugate may be manufactured, for example, by lyophilizing mixing, dissolving, emulsifying, encapsulating, or entrapping a conjugate of this disclosure. Pharmaceutical compositions may also include conjugates of this disclosure in a free-base form or a pharmaceutically acceptable salt form.
[0210] As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0211] In embodiments, the phrase “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
[0212] In embodiments, the term “salt” or “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, /?-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.
[0213] Methods for formulation of the conjugates may include formulating any of the conjugates with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions may include, for example, powders, tablets, dispersible granules and capsules, and in some aspects, the solid compositions further contain nontoxic, auxiliary substances, for example wetting or emulsifying agents, pH buffering agents, and other pharmaceutically-acceptable additives. Alternatively, the conjugates may be lyophilized or in powder form for re-constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
[0214] Pharmaceutical compositions of the conjugates provided herein may comprise at least one active ingredient (e.g., a conjugate and optionally other agents). The active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug-delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
[0215] Pharmaceutical compositions may comprise more than one active compound (e.g., a compound, salt or conjugate and other agents) as necessary for the particular indication being treated. The active compounds may have complementary activities that do not adversely affect each other. For example, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth-inhibitory agent, anti-hormonal agent, anti-angiogenic agent, or cardioprotectant. Such molecules may be present in combination in amounts that are effective for the purpose intended.
[0216] The compositions and formulations may be sterilized. Sterilization may be accomplished by filtration through sterile filtration.
[0217] Compositions of this disclosure may be formulated for administration as an injection. Non-limiting examples of formulations for injection may include a sterile suspension, solution or emulsion in oily or aqueous vehicles. Suitable oily vehicles may include lipophilic solvents or vehicles such as fatty oils or synthetic fatty acid esters, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension. The suspension may also contain suitable stabilizers. Injections may be formulated for bolus injection or continuous infusion. Alternatively, a composition of this disclosure may be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0218] For parenteral administration, the conjugates may be formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles may be inherently non-toxic, and non-therapeutic. Vehicles may be water, saline, Ringer’s solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability (e.g., buffers and preservatives).
[0219] Sustained-release preparations may also be prepared. Examples of sustained- release preparations may include semipermeable matrices of solid hydrophobic polymers that may contain the compound, salt or conjugate, and these matrices may be in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices may include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and y ethyl -L-glutamate, non-degradable ethylenevinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPO™ (z.e., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly -D-(-)-3 -hydroxybutyric acid.
[0220] Pharmaceutical formulations may be prepared for storage by mixing a conjugate of this disclosure with a pharmaceutically acceptable carrier, diluents, excipient, or a stabilizer. This formulation may be a lyophilized formulation or an aqueous solution. Acceptable carriers, diluents, excipients, or stabilizers may be nontoxic to recipients at the dosages and concentrations used. Acceptable carriers, diluents, excipients, or stabilizers may include buffers, such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives, polypeptides; proteins, such as serum albumin or gelatin; hydrophilic polymers; amino acids; 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; or non-ionic surfactants or polyethylene glycol.
[0221] In some embodiments, an aqueous formulation of a conjugate provided herein, such as for subcutaneous administration, has a pH from 4-5.2. The aqueous formulation may comprise one or more excipients, such as one or more buffering agents, one or more lyoprotectants, and the like. In some embodiments, the pH of the formulation is from 4-5.1, 4.1- 5.1, 4.2-5.1, 4.3-5.1, 4.4-5.1, 4.5-5.1, 4-5, 4.1-5, 4.2-5, 4.3-5, 4.4-5, or 4.5-5. In some embodiments, the formulation comprises at least one buffer. In various embodiments, the buffer may be selected from histidine, citrate, aspartate, acetate, phosphate, lactate, tromethamine, gluconate, glutamate, tartrate, succinate, malic acid, fumarate, a-ketoglutarate, and combinations thereof. In some embodiments, the buffer is at least one buffer selected from histidine, citrate, aspartate, acetate, and combinations thereof. In some embodiments, the buffer is a combination of histidine and aspartate. In some embodiments, the total concentration of the buffer in the aqueous formulation is at least 0.01 mM, 0.1 mM, 1 mM, 5 mM, or 10 mM. In some embodiments, the total concentration of the buffer in the aqueous formulation is between 10 mM and 40 mM. In some embodiments, the total concentration of the buffer in the aqueous formulation is between 15 mM and 30 mM. In some embodiments, the total concentration of the buffer in the aqueous formulation is between 15 mM and 25 mM. In some embodiments, the total concentration of the buffer in the aqueous formulation is 20 mM or about 20 mM.
[0222] In some embodiments, the aqueous formulation comprises at least one lyoprotectant. In some such embodiments, the at least one lyoprotectant is selected from sucrose, arginine, glycine, sorbitol, glycerol, trehalose, dextrose, alpha-cyclodextrin, hydroxypropyl beta-cyclodextrin, hydroxypropyl y-cyclodextrin, proline, methionine, albumin, mannitol, maltose, dextran, and combinations thereof. In some embodiments, the lyoprotectant is sucrose. In some embodiments, the total concentration of lyoprotectant in the aqueous formulation is 3-12%, such as 5-12%, 6-10%, 5-9%, 7-9%, or 8%. [0223] In some embodiments, the aqueous formulation comprises at least one surfactant. Exemplary surfactants include polysorbate 80, polysorbate 20, poloxamer 88, and combinations thereof. In some embodiments, the aqueous formulation comprises polysorbate 80. In some embodiments, the total concentration of the at least one surfactant is 0.01%-0.1%, such as 0.01%-0.05%, 0.01%-0.08%, or 0.01%-0.06%, 0.01%-0.04%, 0.01%-0.03%, or 0.02%.
[0224] In some embodiments, the concentration of the conjugate in the aqueous formulation is 1 mg/mL-200 mg/mL, such as 10 mg/mL-160 mg/mL, 10 mg/mL-140 mg/mL, 10 mg/mL-120 mg/mL, 20 mg/mL-120 mg/mL, or 30 mg/mL-120 mg/mL, or 40 mg/mL-120 mg/mL, or 40 mg/mL-100 mg/mL. In some embodiments, the concentration of the conjugate in the aqueous formulation is 10 mg/mL-140 mg/mL or 40 mg/mL-140 mg/mL.
[0225] Pharmaceutical formulations may have conjugates of this disclosure with an average ratio of the drug (exatecan) to anti-PSMA antibody or antigen binding fragment thereof (referred to herein as a drug-to-antibody ratio, or DAR) that ranges from 1 to about 20, from about 1 to about 16, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 5, from 1 to about 3, from about 2 to about 16, from about 2 to about 10, from 2 to about 8, from 2 to about 6, from 2 to about 5, from 2 to about 4, from about 3 to about 16, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, or from about 3 to about 5, from about 4 to about 16, from about 4 to about 10, from about 4 to about 8, from about 5 to about 10, from about 5 to about 8, from about 5 to about 8, from about 6 to about 10, from about 6 to about 8, wherein the drug is exatecan. In some embodiments, the average ratio of the exatecan to anti- PSMA antibody or antigen binding fragment thereof of conjugates in a pharmaceutical formulation may range from 1 to 20, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 5, from 1 to 3, from 2 to 8, from 2 to 6, from 2 to 5, from 2 to 4, from 3 to 8, from 3 to 6, from 3 to 5, from 4 to 8, or from 6 to 8, wherein the drug is exatecan. In some embodiments, the average ratio of the conjugate is 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, 6 or about 6, 7 or about 7, or 8 or about 8.
Methods of Treatment
[0226] The present disclosure provides methods of treating or preventing diseases or conditions (e.g., PSMA expressing cancers, also referred to PSMA+ cancers) comprising administering to a subject in need thereof an effective amount of anti-PSMA conjugates or compositions thereof disclosed herein. Similarly, the present disclosure provides use of anti- PSMA conjugates or compositions thereof disclosed herein in the manufacture of a medicament for treating or preventing diseases or conditions, such as a PSMA expressing cancer.
[0227] Anti-PSMA conjugates, compositions thereof, and methods of this disclosure are useful for treatment or prevention of disease in a plurality of subject species including humans, mammals, non-human mammals, non-human primates, dogs, cats, rodents, mice, hamsters, cows, birds, chickens, fish, pigs, horses, goats, sheep, rabbits, guinea pigs and any combination thereof. In preferred embodiments, anti-PSMA conjugates, compositions thereof, and methods of this disclosure are useful for treatment or prevention of a disease in a human.
[0228] In some embodiments of the methods of the disclosure, a subject in need of treatment has been diagnosed with the disease or condition. In some embodiments, the subject has been treated with another therapy. In some embodiments, the subject is resistant to another therapy or has relapsed following administration of another therapy, and therefore, is in need of treatment by providing or administrating to the subject one or more of an anti-PSMA conjugate or a composition thereof of this disclosure.
[0229] In some embodiments of the methods of this disclosure, a therapeutically effective amount of one or more of an anti-PSMA conjugate or composition thereof of this disclosure is administered to a subject in need thereof. In practicing the methods described herein, therapeutically effective amounts of the anti-PSMA conjugates and pharmaceutical compositions thereof can be administered to a subject in need thereof, often for treating or preventing a condition or progression thereof. The anti-PSMA conjugates and pharmaceutical compositions thereof of this disclosure can affect the physiology of the subject, such as the immune system, an inflammatory response, or other physiologic affect. A therapeutically effective amount can vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.
[0230] As used herein, the term “effective amount” or “effective dose” refers to a quantity of a binding protein conjugate or composition thereof sufficient to achieve a desired (e.g., beneficial) effect in a subject being treated with that compound, conjugate, or composition thereof, such as an amount sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner, delaying worsening of a progressive disease in a statistically significant manner, or preventing onset of additional associated symptoms or diseases in a statistically significant manner, or any combination thereof. In certain embodiments, an effective amount of a binding protein conjugate or composition thereof is an amount sufficient to inhibit or treat the disease with minimal to no toxicity in the subject, excluding the presence of one or more adverse side effects. An effective amount or dose can be administered one or more times over a given period of time. An effective amount or dose can depend on the purpose of the treatment and can be ascertainable by one skilled in the art based on a subject’s needs. When referring to an individual active ingredient, administered alone, an effective amount or dose refers to that ingredient alone. When referring to a combination, an effective amount or dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously.
[0231] Treatment,” “treat,” or “treating” refer to an intervention that leads to any observable beneficial effect of the treatment or any indicia of statistically significant success in the treatment or amelioration of the disease or condition, such as ameliorating a sign, symptom, or progression of a disease or pathological condition. The beneficial effect can be evidenced by, for example, a reduction, delayed onset, or alleviation of the severity of clinical symptoms of the disease in a subject, a reduction in the frequency with which symptoms of a disease are experienced by a subject, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease.
[0232] A prophylactic treatment meant to “prevent” a disease or condition (e.g., tumor formation or growth, in a subject or patient) is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs, for the purpose of decreasing the risk of developing pathology or further advancement of the early disease. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual. A prophylactic treatment can mean preventing recurrence of a disease or condition in a patient that has previously been treated for the disease or condition, e.g., by preventing relapse or recurrence of cancer.
[0233] PSMA expressing cancers can be classified as having a low, intermediate or heterogeneous, or high expression level of PSMA. A low expression level refers to a cancer having less than 1 x 103 PSMA ligands per cell; an intermediate or heterogeneous expression level refers to a cancer having from 1 x 103 to 1 x 104 PSMA ligands per cell; a high expression level refers to a cancer having greater than 1 x 104 PSMA ligands per cell. Expression levels for purposes of classifying the cancers as either low, intermediate or heterogeneous, or high PSMA expression levels is determined by flow cytometry methods known to those of ordinary skill in the art and demonstrated herein in the examples. [0234] PSMA expressing cancers that may be treated with the conjugates and compositions of the disclosure include prostate cancer, salivary gland cancer, thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer. In some embodiments, the PSMA expressing cancer is metastatic. In some embodiments, the PSMA expressing cancer is metastatic prostate cancer. In some embodiments, the PSMA expressing cancer is metastatic castrate resistant prostate cancer.
[0235] In some embodiments, the PSMA-expressing cancer expresses an intermediate level of PSMA. In other embodiments, the PSMA-expressing cancer expresses a high level of PSMA. In other embodiments, the PSMA-expressing cancer expresses a heterogeneous level of PSMA. In various embodiments, the tumor to plasma ratio of free exatecan in the subject is within 30% of the tumor to plasma ratio of free exatecan in a subject having a high PSMA - expressing cancer, preferably wherein the tumor to plasma ratio of free exatecan is within 25%, 20%, 15%, 10% or 5%. For example, in some embodiments, the tumor to plasma ratio is higher in the subject having a PSMA-expressing cancer that expresses an intermediate or heterogeneous level of PSMA as compared to the tumor to plasma ratio in a subject having a PSMA-expressing cancer that expresses high level of PSMA.
[0236] Conjugates and compositions of the disclosure may be administered to a subject by any route, including systemic, intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrasternal, intratumoral, intraspinal, intrathecal, or intraperitoneal injection or infusion.
[0237] The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The phrases “intravenous administration” and “administered intravenously” as used herein refer to injection or infusion of a conjugate into a vein of a subject. The phrases “subcutaneous administration,” “subcutaneously administering,” or the like refer to administration of a conjugate into the subcutis of a subject. For clarity, a subcutaneous administration is distinct from an intratumoral injection into a tumor or cancerous lesion located in the subcuta. [0238] Conjugates and compositions of the disclosure may be administered to a subject in one or more doses, one or more injections/infusions, one or more cycles, or one or more administrations. Conjugates and compositions of the disclosure may be administered to a subject daily, weekly, or monthly. Conjugates and compositions of the disclosure may be administered to a subject once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29 or 30 days. Conjugates and compositions of the disclosure may be administered to a subject once every 1, 2, 3, or 4 weeks. Conjugates and compositions of the disclosure may be administered to a subject once every 1-4 weeks, every 1-3 weeks, or every 1-2 weeks. Conjugates and compositions of the disclosure may be administered to a subject once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
[0239] Conjugates and compositions of this disclosure may be administered to a subject in one or more doses, wherein each dose or the total dose per treatment cycle may comprise between 0.1 mg/kg and 100 mg/kg, inclusive of the endpoints. Conjugates and compositions of this disclosure may be administered to a subject in one or more doses, wherein each dose or the total dose per treatment cycle may comprise between about 0.1 mg/kg and about 100 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise between 0.1 mg/kg and 30 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise between 1 mg/kg and 30 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise between about 1 mg/kg and about 15 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise between 1 mg/kg and 10 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise between about 1 mg/kg and about 10 mg/kg, inclusive of the endpoints. In some embodiments, each dose or the total dose per treatment cycle may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29 or 30 mg/kg. In some embodiments, each dose or the total dose per treatment cycle may comprise 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mg/kg or any number of mg/kg in between. In some embodiments, each dose or the total dose per treatment cycle may comprise about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, about 30 mg/kg or any number of mg/kg in between. In some embodiments, the treatment cycle comprises one or more treatment administrations and one or more periods of observation. In some embodiments, the treatment cycle comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In some embodiments, the treatment cycle comprises at least 1, 2, 3, or 4 weeks. In some embodiments, the treatment cycle comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
[0240] Conjugates and compositions of this disclosure may be administered to a subject in combination with an additional therapy or therapeutic agent. In some embodiments, an additional therapy or therapeutic agent comprises surgery, a hormone therapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, a molecularly targeted therapeutic agent, thermotherapy, radiation therapy, or a vaccine.
[0241] Hormone therapy, also referred to as androgen suppression therapy, for prostate cancer aims to reduce the levels of androgens in the body or stop them from fueling prostate cancer growth. In some embodiments, hormone therapy comprises androgen deprivation therapy, anti -androgen therapy (also referred to androgen receptor antagonists), and estrogen therapy. Androgen deprivation therapy targets production of androgens. In some embodiments, androgen deprivation therapy comprises a luteinizing hormone-releasing hormone (LHRH) agonist, such as leuprolide, goserelin, triptorelin, or leuprolide mesylate; a LHRH antagonist, such as degarelix or relugolix; a CYP17 inhibitor, such as abiraterone; or ketoconazole. In some embodiments, anti -androgen therapy comprises bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, proxalutamide, cimetidine, or topilutamide.
[0242] Chemotherapeutic agents may include erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FTJ (5 -fluorouracil), 5-FU (5 -fluorouracil) leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafamib (SCH 66336, Zokinvy™, Eiger BioPharmaceuticals), sorafenib (BAY43- 9006, Bayer Labs.), and gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; antifolate antineoplastic such as pemetrexed (ALIMTA®, Eli Lilly), aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics, calicheamicin, calicheamicin gamma II and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins, cacti nomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino - doxorubicin and deoxydoxorubicin), epimbicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodombicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5- FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiandrogens (e.g., enzalutamide) or androgen deprivation therapy; anti -adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2”-tri chlorotri ethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin, nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; D,L-alpha-difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0243] Immunotherapeutic agents include immunostimulatory agents, immune checkpoint inhibitors, antibody therapy, and cellular immunotherapy. Immunotherapeutic agents include naturally occurring molecules or cells (e.g., monoclonal antibodies, TCRs) and non- naturally occurring molecules or cells, such as recombinant or fusion molecules (e.g., bispecific T cell engager). Examples of immunostimulatory agents include cytokines, such as IL-2, IL-12, IL-15, IFNa, IFNy, TNFa; chemokines, such as CCL21; and immunostimulatory oligonucleotides. Examples of immune checkpoint inhibitors include inhibitors targeting PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, PVRIG/CD112R, or any combination thereof. In some embodiments, an immune checkpoint inhibitor may be an antibody, a peptide, an RNAi agent, or a small molecule. An antibody specific for CTLA-4 may be ipilimumab or tremelimumab. An antibody specific for PD-1 may be pidilizumab, nivolumab, or pembrolizumab. An antibody specific for PD-L1 may be durvalumab, atezolizumab, or avelumab. Examples of cellular immunotherapy include T cell receptors (TCRs), tumor infiltrating lymphocytes, and chimeric antigen receptors (CARs).
[0244] A cancer vaccine may be a non-antigen specific vaccine (e.g., whole cell vaccine) or an antigen-specific vaccine, such as a protein vaccine, peptide vaccine, protein vaccine, cancer-associated membrane carbohydrate vaccine, mRNA vaccine, nucleic acid vaccine, or antigen-loaded dendritic cell vaccine (e.g., sipuleucel-T). [0245] Radiation therapy includes external beam radiation therapy e.g., conventional external beam radiation therapy, stereotactic radiation, 3 -dimensional conformal radiation therapy, intensity-modulated radiation therapy, volumetric modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, systemic radioisotope therapy, intraoperative radiotherapy, or any combination thereof.
[0246] In some embodiments, the conjugates and compositions of this disclosure may be administered to a subject in combination with an agent targeting other prostate cancer targets, for example, STEAP1, or B7H3.
[0247] The present inventors have discovered that, contrary to conventional wisdom, increased tumor growth inhibition (TGI), even for large tumors (e.g., at least 250 mm3), is obtained when the conjugate of Formula I is administered in two or more doses within a 30-day period instead of administering the same total amount of conjugate in a single larger dose within the same period. Namely, the examples set forth herein demonstrate that fractionating the conjugate dose into two or more smaller doses, and dosing more frequently, results in increased and prolonged TGI compared to administering the same total amount of conjugate in single dose.
[0248] Accordingly, in some embodiments, the present disclosure provides a method for treating a subject having a PSMA expressing cancer, comprising administering to a subject in need thereof a predetermined amount of the conjugate of Formula (I), wherein the predetermined amount is administered to the subject in two or more doses within a 30-day period (i.e., the predetermined amount is split into two or more doses, which are administered separately within the 30-day period). For example, in some embodiments the predetermined amount is administered to the subject in two doses within the 30-day period, the predetermined amount is administered to the subject in three doses within the 30-day period. In other embodiments, the predetermined amount is administered to the subject in more than three doses within the 30-day period.
[0249] In some different embodiment, the two or more doses are administered at a 7 to 15-day interval. For example, in some embodiments the two or more doses are administered at a 7-day interval, 8-day interval, 9-day interval or 10-day interval.
[0250] In other embodiments, the two or more doses are administered within a 20-day period. For example, in some embodiments the predetermined amount is administered in three doses within a 20-day period. [0251] In other embodiments, the administering results in an exatecan tumor to plasma ratio in the subject of at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100: 1, at least 110:1, at least 120: 1, at least 130: 1, at least 140: 1 or at least 150: 1.
[0252] In different embodiments, a period of mean tumor growth inhibition (TGI) in the subject is extended compared to administering the approximately predetermined amount in a single dose. For example, in some embodiments the TGI is extended at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, or at least 400%.
[0253] In other embodiments, the method results in tumor growth inhibition (TGI) in the subject for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days. For example, in some embodiments the TGI is 60%, 70%, 80%, 90% or greater.
[0254] In certain embodiments, the method results in complete inhibition of tumor growth for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days.
[0255] In still other embodiments, the subject has a PSMA expressing cancer with a tumor volume of at least 125 mm3, at least 175 mm3, at least 200 mm3, at least 250 mm3, at least 300 mm3, at least 350 mm3, at least 400 mm3, at least 450 mm3, at least 500 mm3, at least 550 mm3, at least 600 mm3 or at least 650 mm3 prior to administering the two or more doses.
[0256] In some aspects the subject is a human. In other aspects, the PSMA expressing cancer is a cancer described herein, such as prostate cancer, salivary gland cancer, thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer. For example, in some embodiments the subject has prostate cancer, such metastatic prostate cancer or metastatic castrate resistant prostate cancer. Other specific examples include salivary gland cancer.
[0257] In some embodiment the conjugate is administered systemically, and in other embodiments the conjugate is administered intravenously.
[0258] In some embodiments, the method further comprises administering an additional therapy, such as those described herein above. For example, in some embodiments the additional therapy comprises surgery, a hormone therapeutic agent (e.g., anti-androgen therapeutic agent), a chemotherapeutic agent, an immunotherapeutic agent, a molecularly targeted therapeutic agent, thermotherapy, radiation therapy, or a vaccine. For example, in some embodiments the additional therapeutic agent is bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, proxalutamide, cimetidine, or topilutamide, for example enzalutamide. [0259] In certain embodiments, subjects are first screened to determine the PSMA expression level of their cancer, and then a treatment regimen is designed based on the individual subject’s PSMA expression level. Accordingly, in one embodiment is provided a method for treating a PSMA-expressing cancer in a plurality of subjects, the method comprising: determining the PSMA expression level of the cancer in each subject; classifying each subject as having either: a) a low level of PSMA expression; b) an intermediate or heterogeneous level of PSMA expression; c) a high level of PSMA expression; and administering the conjugate disclosed herein, or a pharmaceutical composition of comprising, to a subject classified as having an intermediate or heterogeneous, or high level of PSMA expression.
[0260] In other embodiments of such a method, the method further includes determining an appropriate dose of the conjugate based on the subject’s PSMA expression level.
[0261] In some embodiments, the subject is classified as having an intermediate or heterogeneous level of PSMA expression. In other embodiments, the subject is classified as having a high level of PSMA expression
EXAMPLE 1
PREPARATION OF CONJUGATE ALD101
Cytotoxic Agent
[0262] The payload used in the present invention is a cytotoxic agent, Compound 101. Compound 101 is exatecan, a camptothecin derivative ((15,95)-l-amino-9-ethyl-5-fluoro-9- hydroxy-4-methyl-l,2,3,9,12,15-hexahydro-10Z/,13Z/-benzo[6fe]pyrano[3',4':6,7]indolizino[l,2- Z>] quinoline- 10,13 -di one) .
Figure imgf000056_0001
[0263] Compound 101 can be obtained, for example, by a method described in U.S.
Patent Publication No. US2016/0297890 or other known methods, and the amino group at position 1 can be preferably used as a connecting position to the linker structure. The synthesis and test of the relevant compounds are incorporated herein by reference.
Linker-Payload
[0264] The linker-payload LD101 can be obtained, for example, by a method described in PCT Publication No. WO 2023/280227 or other known methods, and the maleimide group was used as a connecting position to the antibody structure. The synthesis and test of the relevant compounds are incorporated herein by reference. Non-limiting examples of synthesis are incorporated by reference as follows:
Figure imgf000057_0001
LD101
[0265] A Drug-Linker (LD101) containing a cleavable linker attached to exatecan was prepared as follows: Step 1:
Figure imgf000058_0001
101-1 101-2
[0266] A solution of compound 101-1 (650 mg, 0.774 mmol) and N-hydroxysuccinimide (HOSu) (177.98 mg, 1.548 mmol) in anhydrous DCM (8 mL) was stirred at room temperature (r.t.), and then EDCI (296.69 mg, 1.548 mmol) was added. The resulting solution was stirred for another 1 hour at r.t. until liquid chromatography mass spectrometry (LCMS) indicated that all starting amine was consumed and the desired product was detected. The resulting solution was washed with water, the organic layer was collected, and then the water phase was extracted with DCM (10 mL *2). The combined organic layer was dried over sodium sulfate and filtered, concentrated to dryness to yield compound 101-2 (552 mg, 0.589 mmol, 76.12%) as colorless oil and used as such in the next step. LCMS: m/z = 959.4 (M+Na) +;
Step 2:
Figure imgf000058_0002
101-2 101-4
[0267] A solution of compound 101-2 (300 mg, 0.357 mmol) and DIPEA (138.22 mg, 1.071 mmol) in anhydrous DMF (2 mL) was stirred at room temperature, and then compound 101-3 (87.97 mg, 0.357 mmol) was added and the starting amine was suspended in the solution. The resulting mixture was kept stirring at r.t. for another 6 hours. The starting amine dissolved gradually during this period, and the suspension turned into a clear light-yellow solution. The reaction solution was terminated and purified directly by reverse phase liquid chromatography (40 g C18 column, eluting with 0-100%acetonitrile in water with 0.01% TFA over 15 min) to yield compound 101-4 (260 mg, 0.243 mmol, 68.14%) as pale-yellow oil, LCMS ((M-100) /2+H) + = 484.9;
Step 3:
Figure imgf000059_0001
101-4 101-5
[0268] A solution of compound 101-4 (260 mg, 0.243 mmol) in acetonitrile (1.8 mL) was stirred at r.t. and diethyl amine anhydrous (0.2 mL, 1.941 mmol) was added. The resulting solution was stirred at r.t. for 2 hours until the LCMS of the solution showed that most of starting material was consumed. Then the solution was concentrated to dryness and the residue was purified by reverse phase column chromatography (12 g C18 column, eluting with 0-50% acetonitrile in water with 0.01% TFA) to yield the expected fractions of compound 101-5 (170 mg, 0.201 mmol, 82.54%) as pale-yellow oil. LCMS, ESI m/z = 846.6 (M+H)+; retention time (0.01%TFA) = 1.451 min; no UV.
Step 4:
Figure imgf000060_0001
[0269] A clear reaction solution of 101-5 (170 mg, 0.201 mmol), D-glucose (217.08 mg, 1.206 mmol) and acetic acid (1.21 mg, 0.020 mmol) in methanol (5 mL) was heated at 50 °C for 30 min, and then NaCNBHs (75.98 mg, 1.206 mmol) was added. The resulting solution was stirred at 50 °C under N2 for 4 hours. Then additional NaCNBHs (75.98 mg, 1.206 mmol) and D- glucose (217.08 mg, 1.206 mmol) were added and kept stirring at 50 °C for overnight. After stirring for 20 hr, LCMS indicated the reaction was complete. The solvents were evaporated, and the residue was purified by Cl 8 reversed-phase chromatography to yield the desired product 101-6 (106 mg, 0.090 mmol, 44.92%). LCMS, ESI m/z = 537.9 ((M-100) /2+H) +;
Step 5:
Figure imgf000061_0001
[0270] A solution of compound 101-6 (250 mg, 0.213 mmol), HATU (121.45 mg, 0.319 mmol) and DIPEA (82.41 mg, 0.639 mmol) in anhydrous DMF (2 mL) was stirred at r.t. for 5 min, and then compound 101-7 (178.88 mg, 0.213 mmol) was added. The resulting solution was stirred for another 2 hours at r.t. until LCMS indicated a complete reaction. The reaction solution was purified directly by reverse phase liquid chromatography (40 g C18 column, eluting with 0- 70% acetonitrile in water with 0.01% TFA over 15 min) to yield compound 101-8 (270 mg, 0.135 mmol, 63.48%) as a white solid. LCMS, ESI m/z = 666.6 (M/3+H) , 999.2 (M/2+H)+. Step 6:
Figure imgf000062_0001
[0271] A solution of compound 101-8 (120 mg, 0.060 mmol) in TFA (2 mL) was stirred at r.t. for 1 hour. The LCMS of the mixture showed that the reaction was completed, all starting material was consumed, and the desired product (m/z= 633 = 1896/3+H, r.t. 1.501 min) along with the sugar-esterification product (TFA was condensed with hydroxy group in sugar unit, mono-ester with m/z= (1896+96) /2+H=665, R. T. 1.58 min) were formed. The completed reaction solution was condensed to dryness and then redissolved in THF (4 mL) and water (2 mL), and treated with saturated aqueous sodium carbonate solution to adjust the pH to 8-9. The resulting solution was stirred at r.t. for 1 hour to achieve complete hydrolysis. The solution was then neutralized with diluted TFA and condensed. The residue was purified by reverse phase liquid chromatography (C18 column, eluting with 0-25% acetonitrile in water with 0.01% TFA for 15 min) to yield the expected product 101-9 (80 mg, 0.042 mmol, 70.19%) as a white solid after lyophilization. LCMS, ESI m/z = 633.2 (M/3+H) - , 949.2 (M/2+H) ;
Step 7:
Figure imgf000063_0001
[0272] A solution of compound 101-9 (20 mg, 0.011 mmol) and DIPEA (4.08 mg, 0.032 mmol) in anhydrous DMF (1 mL) was stirred at r.t. for 5 min, and then a solution compound 38- 10 (4.88 mg, 0.016 mmol) in anhydrous DMF (1 mL) was added dropwise by syringe over 2 min. The resulting solution was stirred for another 4 hours at r.t. until all starting amine was consumed and the mass of the desired product was detected. The resulting solution was neutralized with formic acid to adjust the pH to 6-7. Then the reaction solution was purified by Prep-HPLC (eluting with gradient with 0.01%TFA over 20 min) to yield Compound LD101 (11 mg, 0.005 mmol, 49.91%) as a white solid. LCMS, m/z = 697.7 (M/3+H) +;
[0273] 1HNMR (400MHz, DMSO-d6) : 5 10.03 (s, 1H) , 8.19-8.11 (m, 2H) , 8.07 (d, J = 8.8 Hz, 1H) , 7.96 (d, J = 7.6 Hz, 1H) , 7.82-7.77 (m, 2H) , 7.66 (d, J = 8.4 Hz, 1H) , 7.60 (d, J = 8.4 Hz, 2H) , 7.36 (d, J = 8.0 Hz, 2H) , 7.32 (s, 1H) , 7.00 (s, 2H) , 6.53 (s, 1H) , 5.99 (t, J = 5.6 Hz, 1H) , 5.45-5.43 (m, 6H) , 5.30-5.24 (m, 3H) , 5.08 (s, 2H) , 4.84-4.74 (m, 2H) , 4.65- 4.49 (m, 4H) , 4.45-4.35 (m, 3H) , 4.27-4.17 (m, 2H) , 4.04-3.95 (m, 2H) , 3.80-3.77 (m, 2H) , 3.71- 3.67 (m, 2H) , 3.62-3.55 (m, 9H) , 3.53-3.43 (m, 44H) , 3.27-3.21 (m, 2H) , 3.16-3.07 (m, 2H) , 3.07-2.93 (m, 6H) , 2.38 (s, 3H) , 2.29 (t, J= 6.4 Hz, 2H) , 2.23-2.13 (m, 2H) , 2 . 13-2.08 (m, 2H) , 2.00-1.82 (m, 4H) , 1.73-1.54 (m, 4H) , 1.54-1.40 (m, 7H) , 1.40-1.30 (m, 4H) , 1.30-1.14 (m, 5H) , 0.90-0.81 (m, 9H) ppm.
Anti-PSMA Conjugate
[0274] To prepare the conjugate ALD101, a solution of 15-25 mg/mL mAh A (anti- PSMA de-immunized HUJ591 antibody having a heavy chain as set forth in SEQ ID NO: 11 and a light chain as set forth in SEQ ID NO: 12) in pH 5.0-6.5 2 mM His-Hac, 150 mM NaCl buffer was reduced by 10 mM TCEP at 30 °C for 180 minutes. TCEP was removed with a Pellicon 3, 30 kD cassette with ULTRACEL® membrane (0.11 m2). 8.5 equivalents ofLDlOl.TFA salt (15 mM) in 20 mM histidine was added to the reduced antibody solution, and the resulting mixture was stirred at 22 °C for 2.5 hours. ALD101 was purified with a Pellicon 3, 30 kD cassette with Ultracel® membrane (0.11 m2) and then filtered with a 0.2 pm sterile filter. This yielded a conjugate with 8 moles of LD101 per mole of antibody.
EXAMPLE 2
ALD101 CHARACTERIZATION AND ADC CONJUGATION SITE ANALYSIS
[0275] Theoretical and measured mass of anti-PSMA antibody and ALD101 by mass spectrometry was obtained. Peptide mapping of anti-PSMA antibody and ALD101 by mass spectrometry confirmed that the N-terminal sequence of the anti-PSMA antibody light chain is DIQMTQSPSSLSTSVGDR and the N-terminal sequence of the anti-PSMA antibody heavy chain is EVQLVQSGPEVK. The C-terminal sequence of the anti-PSMA light chain was confirmed as SFNRGEC in the ADC. The C-terminal sequence of the anti-PSMA heavy chain was confirmed as SLSLSPGK, with the loss of the C-terminal lysine as a modification typically seen in IgGl monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Low ratio unmodified peptide SLSLSPGK was also observed.
[0276] Sample information is provided in Table 2.
Table 2: Sample information for ADC
Figure imgf000065_0001
MW refers to molecular weight
Conjugation Site Confirmation by LC-MS
[0277] In principle, the production of ALD101 involves the mild reduction of the interchain cysteines of the monoclonal antibody intermediate and subsequent chemical conjugation of the linker-payload by maleimide chemistry. Conjugation sites are reported to be involved in the modulation of the in-vivo stability, thus affecting pharmacokinetics and clearance of the ADC. It is therefore important to elucidate the sites of the conjugation for ALD101.
[0278] In this section, the conjugation information of ALD101 samples was distilled from the raw LC-MS/MS data by Lys-C/trypsin sequential digestion and further analyzed. Specifically, the protein was carried denatured with guanidine-hydrochloride (Gdn-HCl) in Tris- HC1 buffer, reduced by dithiothreitol (DTT), and followed by cysteine-alkylation with iodoacetamide (IAM). After sample clean-up, the protein was digested with endoproteinase Lys- C/trypsin sequential digestion to obtain peptides suitable for subsequent analysis. The online LC- MS analysis was performed on an Agilent/UHPLC 1290 system coupled to a Thermo/Q Exactive Orbitrap mass spectrometer. The proteolytic peptides were separated by reversed phase liquid chromatography with an Agilent/Poroshell SB-C18 column, and the Ultraviolet (UV) chromatograms were acquired with the detector set at wavelength of 214 nm. The peptides were further detected by the mass spectrometer operating with full MS scan followed by tandem mass spectrometry (MS/MS) scans.
[0279] Theoretically, there are four inter-chain disulfide bonds in ALD101, corresponding to two sets of four potential conjugation sites: three on the heavy chain and one on the light chain, owing to the symmetric format of the ADC. The predicted conjugation sites and related peptide sequences with Lys-C/trypsin sequential digestion are shown in Table 3. Table 3: Potential conjugation sites and related peptide sequences of ADC by lys-c/trypsin sequential digestion
Figure imgf000066_0002
HC refers to heavy chain; LC refers to light chain.
Positions of conjugation sites in reference to SEQ ID NO: 12 for light chain and SEQ ID NO: 11 for heavy chain
[0280] To identify the conjugation sites, the mass raw data including high resolution MS spectra and MS/MS spectra were analyzed using PMI/Byonic search engine based on the theoretical sequences of HUJ591 mAh intermediate and the linker-payload theoretical mass. The conjugation of LD101 linker-payloads to LC: C214, HC: C218, HC: C224 and HC: C227 were confirmed. The measured masses were in accordance with the theoretical masses with mass differences within 10.0 ppm.
[0281] As shown in the equation below, the conjugation site occupancy ratio (%) at each site was semi -quantitated based on the EIC (Extracted-Ion Chromatograms) of the conjugated peptides against all the existing forms of the corresponding peptides. Peptides identified with conjugation occupancy ratio higher than 3.0% are reported. The conjugation site occupancy (%) is summarized in Table 4. The results indicated that the main conjugation location of LD101 with Antibody A is in the HC-LC inter-chain cysteines.
Figure imgf000066_0001
Conjugation Site Occupancy; is the percentage of linker-payload occupation in the residues i, At,p is the peak area of peptide p when i is attached with LD101 linker-payload in EIC, Ap is the peak area of peptide p in EIC. A schematic representation of ALD101 conjugation sites (LC: C214, HC: C218, HC: C224, and HC: C227) is shown in FIG. 1.
Table 4: Summarized site occupancy of LD101 for each conjugation site
Figure imgf000066_0003
EXAMPLE 3
EVALUATION OF AN ANTIBODY-DRUG CONJUGATE USING AN ANTI-PSMA ANTIBODY DRUG CONJUGATE IN A PROSTATE CANCER MOUSE XENOGRAFT
Materials and Methods:
[0282] Antibody drug conjugate ALD101 was diluted in 20 mM histidine, pH 5.5. For in vitro experiments, ALD101 was diluted in cell culture medium at the desired concentration.
Antibody-Drug Conjugate Solution Preparation
[0283] Formulations were prepared in the biosafety cabinets. ALD101 and control nonbinding ADCs (ADC control) were thawed on ice and kept on ice until they were used to minimize the formation of ADC aggregates. ADC stock solutions were resuspended gently and mixed with cold medium (for in vitro experiments) or cold saline (for in vivo experiments) to prepare the dosing solutions. Formulations were mixed immediately before usage by vortexing.
Cell Lines
[0284] LNCaP, 22Rvl, PC3, and DU145 human PC cell lines were purchased from American Tissue Culture Collection (ATCC, Manassas, VA). The LNCaP-abl human PC cell line was a gift from Prof. Myles Brown (Dana-Farber Cancer Institute, Boston). All cell lines in the laboratory are periodically checked for Mycoplasma spp. infection using the MYCOALERT™ kit (Lonza Group Ltd., Basel, Switzerland), according to the manufacturer’s instructions.
Cell Culture
[0285] LNCaP, 22Rvl, PC3, and DU145 cell lines were cultured in RPMI 1640 medium with L-glutamine (cat# 21875-034, Life Technologies) supplemented with 10% fetal bovine serum (cat # FBS-11A, Capricorn Scientific) and 1% penicillin/ streptomycin (cat# 15140-122, Life Technologies).
[0286] The LNCaP-abl cell line was cultured in phenol red-free-RPMI 1640 (cat# 11835063, Life Technologies) supplemented with 10% charcoal- stripped serum (CSS, PAN- Biotech GmbH, Aidenbach, Germany) and 1% penicillin-streptomycin. Tumor Tissue Single Cell Suspension
[0287] PSMA expression in tumor tissue from CDX mice evaluated for ADC antitumor activity was analyzed by flow cytometry. Tumors were harvested and digested to create a single cell suspension. Tumor tissue was isolated, minced and processed. Briefly, chopped tumor tissue was digested for 30 min to 1 hour at 37 °C on a shaker (FIG. 2) in 2 mL Digestion Buffer composed of RPMI containing 200 U/ml collagenase D from Clostridium histolyticum (cat# 11- 088858001, Roche) and 50 U/mL DNase I solution (cat# 4536282001, Sigma). After enzymatic dissociation, the cell suspension was passed through a 100 pM cell strainer (cat# 11814389001, Roche) to eliminate macroscopic tissue pieces and then resuspended in 2-volume RBC lysis buffer (cat# 11814389001, Roche) for 3 min at room temperature (RT) to eliminate red blood cells. Single cell suspensions were analyzed by flow cytometry.
[0288] All cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.
Flow Cytometry Analysis
[0289] Flow cytometry was performed on tumor tissue (tumor tissue from CDX mice described above) and PC cell lines (cells at a confluency of 85% were removed from the flask with 1 mM EDTA in PBS). Single-cell suspensions of tumor cells were processed for analysis using PBS with 1% FBS, 2 mmol/L EDTA, and fluorochrome-conjugated monoclonal antibodies (mAbs) against PSMA (phycoerythrin-conjugated anti-human PSMA, LNI-17, BioLegend, #342504) or unconjugated PSMA (HUJ591, Wuxi). Secondary antibody was Alexa Fluor® 647- labeled AffiniPure goat anti-human IgG, Fc fragment specific (cat# 109-605-098, Jackson ImmunoResearch). Cells were stained with a live-dead marker to discriminate live cells with Zombie NIR fixable viability dye (BioLegend) and analyzed with a BD LSR Fortessa flow cytometer. PSMA molecules bound per cell were quantified using Quantibrite PE Quantitation Kit (BD Biosciences). Data were analyzed using the FlowJo software.
Confocal Fluorescence Microscopy
[0290] PC cell lines were seeded (l-2xl04 cells) on glass coverslips treated with polylysine (Sigma) in 24-well plates and incubated for 24 hours at 37 °C in 5% CO2 with medium (according to the cell line). Cells were fixed with 4% paraformaldehyde for 10 min at RT and stained with anti-human PSMA mAb (LNI-17, BioLegend or HUJ591, Wuxi) followed by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Life Technologies). Immunofluorescence imaging of cells was performed using a 20-fold magnification 1.25 NA with an inverted CSLM Leica Stellaris 5 confocal microscope.
Cytotoxicity Assay
[0291] Cells were seeded in a 96-well plate at an appropriate density according to the cell line (typically 1000 to 3000 cells per well in 100 pL of appropriate culture media). After an overnight incubation, a concentration series of exatecan (cat# HY- 13631 A, Lucerna AG), ALD101, a control nonbinding ADC, or unconjugated antibody (HUJ591, Wuxi) was added. The maximum concentration tested was 200 nM for each compound. Exatecan alone was diluted 1 :5 by serial dilution, and unconjugated mAb, ALD101, and control nonbinding ADC were diluted 1 : 10 by serial dilution. Cell viability was evaluated after 4 days using an SRB assay for cell density determination, based on the measurement of cellular protein content. Cell monolayers were fixed with 10% (w/v) trichloroacetic acid, stained with 0.057% (w/v) SRB sodium salt, and the stain reconstituted with 10 mM Tris-base buffer pH 10. Absorbance was measured at 510 nm using a microplate reader (Cytation 5 Imaging multi -Readers, Agilent Technologies). The IC50 values were compared with untreated cells or cells treated with controls, and were determined using inhibition dose-response curve fitting (GraphPad Prism 9). Target expression in each cell line was measured by flow cytometry as described above.
Immunohistochemical Staining
[0292] Tumors were harvested and fixed in 10% neutral -buffered formalin (cat# 5701, Thermo Scientific) overnight and embedded in paraffin according to standard protocols. Slides were prepared using consecutive sections. For antigen retrieval, slides were incubated for 60 min in citrate buffer at pH 6 at 98 °C. Slides were incubated with a mouse mAb against PSMA (cat# M3620, Dako) at the dilution of 1 :400. PSMA detection was performed using the Refine Detection DAB Kit (Leica). Protein blocking was performed using Protein-Block solution (cat# X0909, DAKO Agilent Technologies) for 10 mins at RT. Mouse cross-reactivity was minimized using a biotinylated anti-mouse antibody (cat# BP-9200, Vector Laboratories). Immunohistochemistry staining for PSMA was evaluated according to the percentage of tumor cells with nuclear positivity shown by Ki -67 staining (cat# RM-9106-R7, clone SP6, Histocom AG) using an Aperio ImageScope (Leica). Western Blotting and Antibodies for Analysis of Tumor Tissue and Cell Lysates
[0293] Tumor tissues (25-30 mg) or cell pellets from cell lines were lysed with RIP A buffer supplemented with cocktail phosphatase inhibitors (cat# 4906845001, Roche) and protease inhibitors (cat# 5892953001, Roche). Whole protein lysate was separated on 8-12% SDS-polyacrylamide gels and transferred onto a PVDF membrane (cat# 88518, Thermo Fisher Scientific). The membranes were incubated overnight at 4 °C with primary antibodies, anti- GAPDH (cat# sc-47724, Santa Cruz) and anti-PSMA (cat# OTI3H5, Bio-Techne). Detection was done using secondary antibodies (anti -rabbit IgG HRP, cat# W401B and anti -mouse IgG HRP, cat# W402B, Promega). The protein bands were visualized using the western bright quantum reagent (cat# K-12042-D20, Advansta) and quantified using the Fusion Solo IV LBR system (Witec AG).
Animal Experiments
[0294] All animal experiments were carried out according to the protocol approved by the Swiss Veterinary Authority /Board (TI-10-2010 and TI-42-2018) and received approval by the Ethical Committee of the Institute of Oncology Research.
[0295] All in vivo studies used 6-8-week-old male NOD-Raglnull IL2rgnull (NRG) mice.
Mouse Castration, Tumor Inoculation, Monitoring, and Data Collection
[0296] To evaluate antitumor activity, PSMA expression, and PK parameters by flow cytometry, a CDX model was generated using male mice (NRG mouse model) aged 6 to 8 weeks. Mice were castrated following a specific SOP, and 7 to 10 days later, a single preparation of 2xl06 LNCaP-abl cells was injected subcutaneously into the mouse flank using 1 : 1 Matrigel® Matrix (cat# 354234, Corning) with PBS. The single cell suspension was kept on ice until injection. Cells at low passage number in culture (not more than 20 passages in culture) were used, after Mycoplasma screening. Sporadic cases of renal masses contiguous with the subcutaneous tumor at the injection site were observed. Throughout the experiments, tumor volumes were measured three times per week in two dimensions using a caliper. Tumor growth was recorded using a digital caliper, and the tumor volume (TV), expressed in mm3, was calculated by the formula: length x (width)2/2. Randomization
[0297] Randomization was performed for antitumor evaluation when the mean tumor volume reached approximately 125-175 mm3. The mice were randomized according to their tumor volume to ensure that the tumor volume in different groups was similar. The date of randomization was denoted as Day 0 and corresponds to the treatment administration day per study design.
ADC Administration and Antitumor Evaluation
[0298] Xenograft monitoring started 1 week after PC cell injection and was performed three times per week (FIG. 3). Tumor volumes were measured three times per week. As soon as tumors reached a volume of between 125 to 175 mm3 (approximately 2 weeks after injection), the mice were randomly assigned to the treatment groups or vehicle and treated as described in Table 5 and Table 6.
Table 5: ADC treatment plan to evaluate antitumor efficacy in an LNCaP-abl CDX model
Figure imgf000071_0001
1 Single dose; intravenous administration
Table 6: ADC treatment plan to evaluate antitumor efficacy at a higher ADC dose in an LNCaP-abl CDX model
Figure imgf000071_0002
1 Single dose; intravenous administration
[0299] After ADC treatment, xenograft growth was monitored at 24, 48 and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -800 to 1000 mm3, and animals were sacrificed. Tumor tissue was then collected from each mouse for formalin-fixed paraffin- embedded (FFPE) blocks for immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry.
Results
In Vitro Experiments
PSMA Expression in-Prostate Cancer Cell Lines
[0300] PSMA has emerged as a new treatment option for patients with metastatic castration-resistant prostate cancer (mCRPC). PC cell lines are classified based on androgen receptor (AR) signaling and neuroendocrine (NE) marker expression (Sayar et al, 2023, JCI Insight 8:el62907). Phenotypic characterization of PC cell lines used in the study (Table 7) was performed by flow cytometry analysis and Western blotting to evaluate PSMA expression. PSMA expression on PC cell lines by flow cytometry is shown in FIG. 4. Quantification of PSMA molecules bound per cell by flow cytometry is shown in FIG. 5 and immunoblotting in FIG. 6. Comparison of fluorochrome-conjugated antibody and unconjugated anti-human PSMA HUJ591 by flow cytometry is shown in FIG. 7 and immunofluorescence in FIG. 8. In vitro cytotoxicity assays with exatecan and ALD101 in PC cell lines are shown in FIG. 9 and FIG.
10, respectively. The LNCaP-abl mCRPC cell line was amongst the highest PSMA expressors in the panel of 5 PC cell lines and was used to develop a human CDX mCRPC mouse model. ALD101 exhibited high cytotoxic activity in vitro in LNCaP-abl cells (IC50 =0.13 nM).
Table 7: Characteristics of the PC cell lines used in the in vitro study
Figure imgf000072_0001
Prostate cancer (PC) cell lines used in the study are commercially available (except LNCaP-abl, gift from Prof. Myles Brown (Dana-Farber Cancer Institute, Boston) with detailed characteristics of tumor subtype, tumorigenicity, origin of the cells, and androgen receptor (AR) expression. NEPC refers to neuroendocrine prostate cancer
In Vivo Tumor Growth Inhibition Experiments
PSMA Expression in Inoculated Tumor Cells
[0301] Fluorescence-activated cell sorting (FACS) analysis before tumor cell injection into mice confirmed that LNCaP-abl cells expressed high levels of PSMA (FIG. 11). Cells at low passage number in culture (not more than 20 passages in culture) were used for the CDX mouse model.
Antitumor Activity of a single dose of ALD101 3 and 10 mg/kg
[0302] Tumor growth curves (FIG. 12) and mouse body weight (FIG. 13) for each treatment group from treatment start until study termination are shown.
Effect of a single dose of ALD101 3 and 10 mg/kg on PSMA Expression on Tumor Cells
[0303] PSMA expression was determined in tumor tissue from castrated LNCaP-abl CDX mice after treatment with ALD101 (upon tumor regrowth) (FIGS. 14A-14B).
Antitumor Activity of a single dose of ALD101 10 and 15 mg/kg
[0304] The antitumor activity of 10 mg/kg and 15 mg/kg ALD101 were compared in the LNCaP-abl CDX model (FIG. 15). ALD101 showed a strong dose-dependent antitumor effect in vivo in the LNCaP-abl castrated mCRPC mouse model, with tumor regression and persistent inhibition of tumor growth for about 35 days after a single dose of 10 mg/kg and about 45 days after a single dose of 15 mg/kg.
Tolerability
[0305] Overall, treatment was well-tolerated by all CDX mice given single doses of 3 mg/kg, 10 mg/kg, or 15 mg/kg ALD101. No animal deaths were reported during the study. Mouse weight after 10 mg/kg and 15 mg/kg ALD101 treatment in an LNCaP-abl CDX model is shown in FIG. 16.
EXAMPLE 4
DISTRIBUTION OF EXATECAN AS FREE PAYLOAD OF A PROSTATE SPECIFIC MEMBRANE
ANTIGEN-TARGETED ANTIBODY-DRUG CONJUGATE ALD101 IN A PROSTATE CANCER
XENOGRAFT MODEL
[0306] The aim of this study is to analyze the distribution of free exatecan in tumor tissue and systemic exposure of exatecan after the administration of different doses of ALD101. The method for analyzing free exatecan, based on a published method (Oguma et al, 2005, Chromatogr B Analyt Technol Biomed Life Sci 818:249-256), has been developed and validated previously, the methods for which are incorporated herein by reference.
Materials and Methods:
Table 8: Exatecan
Figure imgf000074_0001
Table 9: [2Hs]-Exatecan (internal standard)
Figure imgf000074_0002
Table 10: Solvents and reagents
Figure imgf000075_0001
Table 11: Equipment used for method development
Figure imgf000075_0002
Antibody-Drug Conjugate, Prostate Cancer Cell Line, and Animal Experiments
[0307] Antibody drug conjugate ALD101 was diluted in 20 mM histidine, pH 5.5. See Example 3 for the description of the ADCs used (ALD101 and non-binding ADC control), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
Antibody-Drug Conjugate Administration and Pharmacokinetic Analyses
[0308] Xenograft monitoring started 1 week after injection of LNCaP-abl PC cells and was performed 3 times per week. Tumor volumes were measured 3 times per week. As soon as tumors reached a volume of between 200 to 250 mm3 (approximately 2 weeks after injection), the mice were randomly assigned to the treatment groups or vehicle, and treated as described in Table 12.
[0309] Mice were sacrificed 24 hours after ADC treatment administration. Tumor tissue, liver tissue, and blood were collected from each mouse for further analysis. Plasma was collected in heparinized tubes and stored frozen, and tumor, and liver tissue was snap-frozen. Plasma and tissue samples were stored at -20°C.
Table 12: ADC treatment plan for pharmacokinetic analysis
Figure imgf000076_0001
1 Singl dose; intravenous administration
Sample Preparation for Quantitative Determination of Free Exatecan in Mice Plasma and Tissue
[0310] Prior to analysis, plasma was thawed at room temperature (RT). Tissue samples were weighed (frozen) and 1 gram was homogenized in 9 mL of PBS to obtain a 1 :9 (w/V) homogenate and immediately processed for LC-MS/MS analysis.
[0311] Ten microliters of [2Hs]-exatecan (500 ng/ml) were added as an internal standard (IS) to 100 pL of plasma or to 200 pL of tumor or liver homogenate prepared 1 :9 (w/V) in PBS. Samples were vortexed and 500 pL methanol and 0.1% formic acid was added to precipitate proteins. Samples were centrifuged at 15000 ref for 10 min at 4°C. The supernatants were transferred into clean tubes and evaporated under gentle N2 flux at 37 °C. The residues were reconstituted in 200 pl of a solution of [9: 1 ratio of (water + 0.1% formic acid: methanol + 0.1% formic acid)], then vortexed and centrifuged at 15000 ref for 10 min at 4 °C. Supernatants were transferred into an autosampler glass vial and a 5 pL sample was injected into the HPLC-MS/MS system (see Calibration Curve and Quality Control Section).
Calibration Curve and Quality Control
[0312] The calibration curve was built with a blank plasma or homogenized tissue sample from mice, a zero-blank plasma or tissue, 1 sample at the lower limit of quantification concentration level (LLOQ), followed by 6 control plasma or tissue samples spiked with 10 pL of different working solutions (Table 8) to obtain the following final concentrations: 0.01, 0.02, 0.075, 0.1, 1, 5 and 10 ng exatecan/sample.
[0313] Quality controls (QCs) were prepared by spiking control plasma and homogenized tissue samples with 10 pL of different working solutions (Table 9) to obtain the following final concentrations: 0.05, 2.5, and 7.5 ng exatecan/sample. Chromatography and Mass Spectrometry Assay
[0314] A validated HPLC-MS/MS method was used to determine exatecan concentrations.
Chromatographic Separation
[0315] Reversed-phase chromatography was performed under gradient conditions with separation on a Kinetex EVO Cl 8 (2.6 pm, 110A, 2.1 x 100 mm) Phenomenex column, coupled with a guard column (2.6 pm, 2.1 x 4 mm) under the following conditions:
Injection volume: 5 pL
Injector temperature: 10°C
Flow rate: 0.2 mL/min
Detection: MS/MS
Column temperature: 30°C
Mobile phase /gradient: MP-A: H2O + 0.1% HCOOH
MP-B: CH3OH + 0.1% HCOOH
Table 13 Chromatographic conditions
Figure imgf000077_0001
Mass Spectrometry: Analyte Detection and Quantification
[0316] Analyte detection and quantification was carried out by MS/MS using a TSQ Altis with the following mass transitions and conditions:
Ion transfer tube temperature: 325 °C
Vaporizer temperature: 275 °C
Ionization mode: Positive
Ion spray voltage: 5 kV
Table 14 Mass Spectrometry Data
Figure imgf000077_0002
Figure imgf000078_0001
IS, internal standard
Data Processing
[0317] Analytical data were acquired using Chromelion 7.3 chromatography data system (CDS) software (ThermoFisher Scientific). Peak area ratios from calibration samples (area of analyte peak divided by area of IS peak) were used to calculate the intercept and the slope of the calibration curve by the weighted least squares method. Calibration curve parameters were calculated using an appropriate weighing factor (l/x2). The goodness of fit of the linear regression was assessed using the coefficient of determination R2 and the back-calculated concentrations of the individual calibration standards.
Statistical Analysis
[0318] Numbers of samples and mice used in each experiment are indicated in figure legends. Data are presented as means ± SDs. P values were calculated by using the Student’s t- test. Statistical analysis was performed with GraphPad Prism software (GraphPad Software, La Jolla, CA). Statistical significance was established as a P value of less than 0.05.
Results
Free Exatecan Distribution in Plasma and Tissue at 3 and 10 mg/kg ALD101
[0319] The distribution of exatecan in tumor bearing mice treated with a single dose of ALD101 (3 mg/kg or 10 mg/kg) or nonbinding control ADC (3 mg/kg or 10 mg/kg) and euthanized 24 hours after treatment, was determined. Plasma, tumor tissue and liver tissue were analyzed by HPLC-MS/MS. The distribution of free exatecan in tumor tissue and liver tissue was significantly higher with ALD101 than with nonbinding control ADC (FIGS. 17A and 17C). In plasma, free exatecan levels were low and comparable at both doses (FIG. 17B), assuring equivalent low systemic exposure. This results in an extremely favorable tumor/plasma ratio (around 80 at both doses) thus improving the therapeutic index of the therapy (FIG. 17D). When using the nonbinding control ADC, the drug concentration in tumor and the tumor/plasma ratio are significantly lower (FIG. 17A and FIG. 17D). Free Exatecan Distribution in Plasma and Tissue at 10 and 15 mg/kg ALD101
[0320] The experiment was repeated to analyze the distribution of exatecan in tumorbearing mice treated with a single dose of ALD101 (10 mg/kg or 15 mg/kg) or nonbinding control ADC (15 mg/kg) and euthanized 24 hours after treatment. The experiment confirmed that the ALD101 was found in the tumor target tissue (FIG. 18A) with a relatively low systemic exposure (FIG. 18B) as highlighted by the extremely favorable tumor/plasma ratio (FIG. 18D). Of note, the higher ADC dose resulted in a significant increase in tumor drug concentration.
EXAMPLE 5
ANTITUMOR ACTIVITY ACCORDING TO TUMOR SIZE PRIOR TO TREATMENT WITH A SINGLE DOSE OF 10 MG/KG OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT
MODEL (LNCAP-ABL)
[0321] This study was conducted according to the general procedure illustrated in FIG.
19 to evaluate the antitumoral activity of a single dose of ALD101 according to the tumor size prior to treatment in a castrated PC cell-line derived xenograft (CDX) immunodeficient NRG or NSG mouse model (LNCaP-abl) using pooled data from 4 studies. Specifically, this example illustrates the relationship between in vivo antitumoral efficacy of a single administration of ALD101 at 10 mg/kg and the size of the tumor at the time of ALD101 treatment in the LNCaP- abl CDX castrated NRG/NSG mouse model in mice treated in one of four studies.
[0322] Antibody drug conjugate ALD101 was diluted in 20 mM histidine, pH 5.5 or in
20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH5.5. Stock solutions of ALD101 were prepared as described in Example 3. Similarly, cell lines, cell culture and animal experiments for this example were the same as described in Example 3. Inoculation parameters for each study were as provided in Table 15.
Table 15: Inoculation Parameters
Figure imgf000079_0001
* Immediately prior to treatment with ALD101 10 mg/kg [0323] After inoculation with tumor cells, the animals were checked daily for 3 days for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormalities. Throughout the experiments, tumor volumes were measured three times per week in two dimensions using a caliper. Tumor growth was recorded using a digital caliper, and the tumor volume (TV), expressed in mm3, was calculated by the formula: length x (width)2/2. Mortality and observed clinical signs were recorded for individual animals. Dosing, tumor volume, and body weight measurements were conducted in a laminar flow cabinet. Tolerability was evaluated by monitoring mortality, body weight, and the appearance of clinical signs of distress for individual animals.
[0324] Depending on the study, the randomization was performed when the mean tumor volume reached approximately either 200, 300 or 600 mm3. Within each experiment the mice were randomized to the study treatment groups according to their tumor volume to ensure that the tumor volume in different groups was similar. The date of randomization was denoted as Day 0 and corresponds to the treatment administration day per study design.
[0325] Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. When tumors reached a volume of approximately 200 to 600 mm3 depending on the experiment (approximately 2-3 weeks after injection), the mice were randomly assigned to the treatment groups or vehicle and treated. The treatment groups analyzed in this study are presented in Table 16.
Table 16: Number of mice (LNCaP-abl CDX) with evaluation of antitumor efficacy following a single dose of ALD101 10 mg/kg
Figure imgf000080_0001
*49 animals were initially treated but 3 animals presenting signs of tumor ulcerations/necrosis were sacrificed and were excluded from grouped analyses, thus 46 animals were included in the analyses.
[0326] After ADC treatment, xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. Following these initial daily observations, CDX tumor growth was monitored three times per week until the tumor reached a maximum volume of 800-1500 mm3 or until the maximum observation period specified by the animal license was reached, at which point the animals were sacrificed. Tumor tissue was then collected from each mouse for formalin-fixed paraffin-embedded (FFPE) blocks for immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry.
[0327] Tumor volume data over time from mice treated with ALD101 10 mg/kg in the 4 studies were pooled and analyzed in 3 groups according to the size of the tumor immediately prior to treatment: below 250 mm3, between 250 and 350 mm3 and above 350 mm3. Data were analyzed for tumor volume measurements for mice with tumor measurements through to 40 days after administration of ALD101 10 mg/kg on Day 0; note that for mice treated in study 4 that received a second dose of ALD101 10 mg/kg on Day 18, only tumor volume data collected up to Day 18 before the second injection were included in the current analysis. The number of mice included per analysis group is presented in Table 16. Note also that animals presenting signs of tumor ulcerations/necrosis were sacrificed and were excluded from grouped analyses.
Results
[0328] FIG. 20 shows tumor growth curves for each pre-treatment tumor size group in CDX NRG mice, from treatment start (Day 0 corresponding to randomization and ALD101 administration) until Day 40.
[0329] This example provides compelling evidence of differences in tumor growth inhibition (TGI) according to tumor size at time of treatment for a single administration of 10 mg/kg ALD101; in smaller tumors up to 250 mm3 prior to treatment, mean TGI of >90% was reported at multiple time points, whereas maximal mean TGI in mice with tumors 250-350 mm3 was 84%, and maximal mean TGI in mice with tumors >250-350 mm3 was 80%.
EXAMPLE 6
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATION (7.5 MG/KG X3 DOSES) OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0330] This study was conducted according to the general procedure illustrated in FIG. 21 to evaluate the activity of multiple dosing of ALD101 in a castrated prostate cancer (PC) CDX mouse model (LNCaP-abl). Antibody drug conjugate ALD101 was diluted 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH5.5. ALD101 solutions were prepared as described in Example 3. Cell lines, cell culture and animal experiments were also as described in Example 3. [0331] Inoculation and tumor growth was monitored according to the general procedures of Example 3. Randomization was performed for antitumor evaluation when the mean tumor volume reached approximately 300 mm3 (range -200-440 mm3). The mice were randomized according to their tumor volume to ensure that the tumor volume in different groups was similar. The date of randomization was denoted as Day 0 (DO) and corresponds to the treatment administration day per study design.
[0332] Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. Tumor volumes were measured three times per week. When tumors reached a volume of approximately 300 mm3 (approximately 2-3 weeks after injection), the mice were randomly assigned to the treatment group or vehicle and treated as described in Table 17.
Table 17: ADC Treatment Plan to Evaluate Antitumor Efficacy with Repeated ADC Doses in an LNCaP-abl CDX Model.
Figure imgf000082_0001
*Note that 3 animals were initially treated with vehicle but 1 animal presenting signs of tumor ulcerations/necrosis was sacrificed and was excluded from grouped analysis
[0333] After ADC treatment, xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm3 or after 82 days, and animals were sacrificed. Tumor tissue was then collected from each mouse for formalin- fixed paraffin-embedded (FFPE) blocks for future immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry.
Results
[0334] Tumor growth curves (presented as mean ± SEM) and mouse body weight (presented as mean ± SD) for each treatment group from treatment start until study termination were measured are shown in FIG. 22 and FIG. 23, respectively.
[0335] ALD101 showed antitumor effects in vivo in a castrated LNCaP-abl CDX immunodeficient NRG model, with a mean tumor volume of 300 mm3 (range -200-440 mm3) at the time of treatment. Major and early tumor inhibition was seen in these mice with large tumors treated with three doses of ALD101 7.5 mg/kg (z.e., lower than the standard dose of 10 mg/kg), with mean TGI >90% lasting 56 days. TGI was durable with complete inhibition of tumor growth lasting for 43 days after the third injection of ALD101 7.5 mg/kg. A single dose of 20 mg/kg has been shown to result in tumor growth inhibition lasting 20 days (see Example 7 - FIG. 25). Fractionating this single dose into three consecutive administrations of ALD101 of 7.5 mg/kg each with a 10-day interval resulted in TGI lasting twice as long (for at least 43 days after the last injection), with complete response for more than 63 days since initiation of treatment.
[0336] Overall, treatment with ALD101 was well tolerated by all CDX mice that received three doses of 7.5 mg/kg ALD101 with an interval of 10 days each, with no body weight loss and no clinical signs of distress observed. No animal deaths were reported during the study.
EXAMPLE 7
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATIONS (10 MG/KG TWICE) COMPARED TO SINGLE ADMINISTRATION (10 OR 20 MG/KG ONCE) OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0337] This example was conducted as according to the general procedure illustrated in FIG. 24 to evaluate the antitumoral efficacy of repeated dosing of ALD101 (10 mg/kg twice at a 10-day interval) compared to single administration (10 or 20 mg/kg once) in this in vivo castrated model. Antibody drug conjugate ALD101 was diluted in 20 mM histidine, pH 5.5 or in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH5.5. ALD101 solutions were prepared as described in Example 3. Cell lines, cell culture, inoculation and animal experiments were also as described in Example 3.
[0338] Randomization was performed for antitumor evaluation when the mean tumor volume reached approximately 200 mm3 (range 104-227 mm3). The mice were randomized according to their tumor volume to ensure that the tumor volume in different groups was similar. The date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design. Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. Tumor volumes were measured three times per week. When tumors reached a volume of approximately 200 mm3 (approximately 2-3 weeks after injection), the mice were randomly assigned to a treatment group or vehicle and treated as described in Table 18. Table 18: ADC treatment plan to evaluate antitumor efficacy at a single versus repeated ADC doses in an LNCaP-abl model
Figure imgf000084_0001
Note that 32 animals were initially treated but 4 animals presenting signs of tumor ulcerations/necrosis were sacrificed and were excluded from grouped analyses, thus 28 animals were included in the analysis.
[0339] After ADC treatment, xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm3 or after 70 days, and animals were sacrificed. Tumor tissue was then collected from each mouse for formalin- fixed paraffin-embedded (FFPE) blocks for immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry.
Results
[0340] Tumor growth curves (presented as mean ± SEM) and mouse body weight (presented as mean ± SD) for each treatment group from treatment start until study termination were measured are presented in FIG. 25 and 26, respectively. Animals presenting signs of tumor ulcerations/necrosis were sacrificed and were excluded from grouped analyses
[0341] ALD101 showed antitumor effects in vivo in a castrated LNCaP-abl CDX immunodeficient NRG model with a mean tumor volume of 200 mm3 (range 104-227 mm3) at the time of treatment. Major tumor inhibition was seen in all mice treated with two doses of ALD101 10 mg/kg, with mean tumor growth inhibition (TGI) > 90% for 14 days, as well as in mice treated with a single dose of ALD101 20 mg/kg with mean TGI > 90% for 9 days. Fractionating the single dose of ALD101 20 mg/kg into two administrations of ALD101 10 mg/kg each, administered with a 10-day interval, resulted in ~2-fold longer TGI (for 27 days compared with 15 days with a single dose at 20 mg/kg).
[0342] Overall, treatment with ALD101 was well tolerated by all CDX mice that received single doses of 10 mg/kg or 20 mg/kg ALD101, as well as by CDX mice that received two doses of ALD101 10 mg/kg with an interval of 10 days, with no body weight loss and no clinical signs of distress observed. No animal deaths were reported during the study. EXAMPLE 8
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATIONS (10 OR 15 MG/KG TWICE) COMPARED TO A SINGLE DOSE OF 20 MG/KG OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0343] This example was performed according to the general procedure outlined in FIG. 27 to demonstrate the antitumoral efficacy of repeated dosing of ALD101 at 10 or 15 mg/kg administered at a 10-day interval compared to a single dose of 20 mg/kg in this in vivo model. Cell lines, cell culture, inoculation and animal experiments were also as described in Example 3.
[0344] Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. ALD101 solutions were prepared as described in Example 3.
[0345] Mice were castrated 10 days before tumor cell injection. A single preparation of 3xl06 LNCaP-abl cells was injected subcutaneously (sc) in the dorsal mouse flank. Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. When tumors reached a volume of approximately 300 mm3 (range -150-500 mm3) approximately 2-3 weeks after injection, the mice were randomized according to their tumor volume and assigned to a treatment group or vehicle and treated as described in Table 19. The date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
Table 19: ADC treatment plan to evaluate antitumor efficacy at a single versus repeated ADC doses in an LNCaP-abl CDX model
Figure imgf000085_0001
[0346] After ADC treatment, xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm3 or after 63 days, and animals were sacrificed. Results
[0347] Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 28 and 29, respectively. Data are presented as mean ± SEM or mean ± SD respectively.
[0348] Major tumor inhibition was seen in these tumor bearing mice treated with two doses of ALD10110 mg/kg, with mean tumor growth inhibition (TGI) > 90% for 31 days, as well as in mice treated with ALD101 15 mg/kg with TGI > 90% for 47 days. TGI was durable lasting for 24 days with a single dose at 20 mg/kg, while administering two doses of 10 mg/kg or 15 mg/kg with a 10-day interval demonstrated TGI lasting at least 42 and 54 days, respectively.
[0349] Fractionating the single dose of ALD101 20 mg/kg into two administrations of ALD101 10 mg/kg each, administered with a 10-day interval, resulted in TGI lasting twice as long (54 days compared with 24 days with a single dose at 20 mg/kg) (FIG. 28).
[0350] Overall, treatment with ALD101 was well tolerated by all CDX mice that received two doses of 10 mg/kg or 15 mg/kg ALD101 with an interval of 10 days, as well as by all CDX mice that received one dose of ALD101 20 mg/kg, with no body weight loss (FIG. 29) and no clinical signs of distress observed. No animal deaths were reported during the study.
EXAMPLE 9
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATION (7.5 MG/KG X2 OR X3 DOSES) OF ALD101 COMPARED TO A SINGLE DOSE OF 7.5 OR 20 MG/KG OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0351] This example was performed according to the general procedure outlined in FIG. 30 to demonstrate the antitumoral efficacy of single or repeated dosing of ALD101 at 7.5 mg/kg administered twice or thrice at 10-day intervals, compared to a single dose of 7.5 or 20 mg/kg in the LNCaP-abl CDX model (using pooled data from 2 studies). Cell lines, cell culture, inoculation and animal experiments were as described in Example 3.
[0352] Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 and prepared according to the general procedure outlined in Example 3.
[0353] Mice were castrated 10 days before tumor cell injection. A single preparation of 2-3xl06 LNCaP-abl cells was injected subcutaneously (sc) in the dorsal mouse flank. Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. When tumors reached a volume of approximately 200 mm3 (range -100-250 mm3) approximately 2-3 weeks after injection, the mice were randomly assigned to a treatment group or vehicle according to their tumor volume to ensure that the tumor volume in different groups was similar, and treated as described in Table 20. The date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
Table 20: ADC treatment plan to evaluate antitumor efficacy at a single versus repeated ADC doses in an LNCaP-abl CDX Model
Figure imgf000087_0001
ulcerations/necrosis were sacrificed and were excluded from grouped analyses (3 in the vehicle group and 2 in the ALDI 01 7.5 mg/kg once and 1 in the ALD101 20 mg/kg once), thus 34 animals were included in the analysis.
[0354] After ADC treatment, xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm3 or after 73 days, and animals were sacrificed.
RESULTS
[0355] Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 31 and 32, respectively. Data are presented as mean ± SEM or mean ± SD respectively.
[0356] Major tumor inhibition was seen in tumor bearing mice treated with two doses of ALD101 7.5 mg/kg, with mean tumor growth inhibition (TGI) > 90% for 33 days, as well as in mice treated with three doses of ALD101 7.5 mg/kg with TGI > 90% for 51 days. TGI (> 80%) was transient, lasting for 10 days with a single dose at 7.5 mg/kg, and lasted 22 days with a single dose at 20 mg/kg. Administering two or three doses of 7.5 mg/kg with a 10-day interval, demonstrated TGI lasting at least 45 and 63 days, respectively.
[0357] Fractionating the single dose of ALD101 20 mg/kg into three administrations of ALD101 7.5 mg/kg each, administered with 10-day intervals, resulted in TGI lasting three times as long (63 days compared to 22 days with a single dose at 20 mg/kg).
[0358] Overall, treatment with ALD101 was well tolerated by all CDX mice that received two or three doses of 7.5 mg/kg ALD101 with an interval of 10 days, as well as by all CDX mice that received one dose of ALD101 7.5 or 20 mg/kg, with no body weight loss and no clinical signs of distress observed. No animal deaths were reported during the study.
EXAMPLE 10
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATION (5 MG/KG X2 OR X3 DOSES) OF ALD101 COMPARED TO A SINGLE DOSE OF 5 MG/KG OR 15 MG/KG OF ALD101 IN A
CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0359] This example was performed according to the general procedure outlined in FIG. 33 to evaluate the antitumoral activity of a single or repeated doses of ALD101 in a castrated PC cell-line derived xenograft (CDX) immunodeficient NSG (NOD.Cg-PrkdcSCID I12rgtmlWjl/SzJ) mouse model (LNCaP-abl). This study was conducted to demonstrate the antitumoral efficacy of single dosing at 5 mg/kg or 15 mg/kg compared to repeated dosing of ALD101 at 5 mg/kg administered twice or thrice at 10-day intervals in this LNCaP-abl CDX model.
[0360] Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 and prepared according to the general procedure outlined in Example 3.
[0361] The LNCaP-abl human CRPC cell line was provided, Mycoplasma-free, by Dr. Catapano, IOR, Bellinzona, Switzerland. Cells were cultured in phenol red-free-RPMI 1640 (ECM0505L, Euroclone) supplemented with 10% charcoal-stripped serum (A3382101, Life Technologies Europe BV) 1% glutamine (X0550100, Microgem), and 1% penicillinstreptomycin (ECB3001D, Euroclone). All cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.
[0362] All studies used 6-8-week-old male immunodeficient NOD.Cg-PrkdcSCID I12rgtmlWjl/SzJ (NSG) mice. [0363] Mouse castration was performed following a specific standard operating procedure (SOP). Twelve to thirteen days after castration, 3xlO6 LNCaP-abl cells in phosphate buffered solution were injected subcutaneously in the right flank of each mouse. LNCaP-abl cells were used at low passage (below 20). Mycoplasma screening was done after the end of the experiment and confirmed the absence of Mycoplasma.
[0364] The growing tumor masses were measured with the aid of a Vernier caliper, and the tumor volume (TV), expressed in mm3, was calculated by the formula: length x (width)2/2.
[0365] Sixteen days after tumor inoculum, when mean TV reached -200 mm3 (range 150-250 mm3) mice were allocated to each experimental group (Table 21) by stratified randomization using TV as the variable. The day of randomization and treatment was denoted as Day 0 (DO).
Table 21: ADC treatment plan to evaluate antitumor efficacy at a single versus repeated ADC doses in an LNCaP-Abl model
Figure imgf000089_0001
[0366] After randomization, mice were treated with a single intravenous dose of ALD101 or vehicle (Table 21). Tumor growth was monitored at least twice a week until reaching a TV of 1500 mm3 or another human end-point (HEP) or after 61 days. Mice that did not reach a HEP were sacrificed at the maximum time points, defined as 3 x median survival time (MST) of the control group. The antitumor activity was expressed as percentage of tumor growth inhibition (%TGI) which is the percentage of mean tumor reduction compared to mean initial tumor volume.
Results
[0367] Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 34 and 35, respectively. Data are presented as mean ± SEM or mean ± SD respectively. [0368] TGI (~ 60%), lasting up to 12 days was seen with a single low dose at 5 mg/kg. Prolonged TGI (~ 70%) up to ~ 21 days was seen in both mice treated with a single dose of ALD101 15 mg/kg as well as in mice treated with two doses of ALD101 5 mg/kg. Major and prolonged TGI (> 70%) lasting up to 45 days was seen in mice treated with three doses of ALD101 at 5 mg/kg.
[0369] Fractionating the single dose of ALD101 15 mg/kg into three administrations of ALD101 5 mg/kg each, administered with a 10-day interval, resulted in TGI lasting more than twice as long (45 days compared with 21 days with a single dose at 15 mg/kg). A low dose of ALD101 (5 mg/kg) can induce major and prolonged TGI > 70% when administered repeatedly at 10-day intervals.
[0370] Overall, treatment with ALD101 was well tolerated by all CDX mice that received two or three doses of 5 mg/kg ALD101 with an interval of 10 days, as well as by all CDX mice that received one dose of ALD101 5 mg/kg or 15 mg/kg, with no body weight loss and no clinical signs of distress observed. No animal deaths were reported during the study.
EXAMPLE 11
ANTITUMOR ACTIVITY OF SINGLE ADMINISTRATION (10 AND 15 MG/KG) OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (22Rvl)
[0371] This example was performed according to the general procedure outlined in FIG. 36 to evaluate the antitumoral activity of a single or repeated doses of ALD101 in a castrated PC cell-line derived xenograft (CDX) immunodeficient NRG (NOD-Raglnull IL2rgnull) mouse model (22Rvl). This study was conducted to demonstrate the antitumoral efficacy of single dosing at 10 mg/kg or 15 mg/kg in a 22Rvl CDX model expressing intermediate and heterogeneous levels of PSMA which differentiates this model from the high PSMA expressing LNCaP-abl CDX model described above.
[0372] Antibody drug conjugate ALD101 was diluted in 20 mM histidine, pH 5.5, and prepared according to the general procedure outlined in Example 3. The 22Rvl cell line was purchased from American Tissue Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium with L-glutamine (cat# 21875-034, Life Technologies) supplemented with 10% fetal bovine serum (FBS-11A, Capricorn Scientific) and 1% penicillin/ streptomycin (cat# 15140-122, Life Technologies). All cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. [0373] All in vivo studies used 6-8-week-old male immunodeficient NOD-Raglnull IL2rgnull (NRG) mice. Mice were castrated following a specific SOP, and 7 to 10 days later, a single preparation of 1.5xl06 22Rvl cells was injected subcutaneously into the mouse flank using 1 : 1 Matrigel® Matrix (cat# 354234, Corning) with PBS. The single cell suspension was kept on ice until injection. Cells at low passage number in culture (not more than 20 passages in culture) were used, after Mycoplasma screening.
[0374] After inoculation with tumor cells, tumor volumes were measured three times per week in two dimensions using a caliper. Tumor growth was recorded using a digital caliper, and the tumor volume (TV), expressed in mm3, was calculated by the formula: length x (width)2/2.
[0375] Randomization was performed when the mean tumor volume reached approximately 200 mm3 (range -100-350 mm3). The mice were randomized according to their tumor volume to ensure that the tumor volume in different groups was similar (Table 22). The day of randomization and treatment was denoted as Day 0 (DO).
Table 22: ADC treatment plan to evaluate antitumor efficacy of a single ADC dose in a 22Rvl CDX model
Figure imgf000091_0001
[0376] After randomization, mice were treated with a single intravenous dose of ALD101 or vehicle (Table 22). Tumor growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of - 800-1000 mm3 or after 40 days, and animals were sacrificed. Tumor tissue was then collected from each mouse for formalin-fixed paraffin- embedded (FFPE) blocks for immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry. The antitumor activity was expressed as percentage of tumor growth inhibition (%TGI) which is the percentage of mean tumor reduction compared to mean initial tumor volume.
[0377] PSMA expression in tumor tissue from CDX mice evaluated for ADC antitumor activity was analyzed by flow cytometry (FIG. 37). Tumors were harvested and digested to create a single cell suspension. Tumor tissue was isolated, minced and processed. Briefly, chopped tumor tissue was digested for 30 min to 1 hour at 37 °C on a shaker in 2 mL Digestion Buffer composed of RPMI containing 200 U/ml collagenase D from Clostridium histolyticum (cat# 11-088858001, Roche) and 50 U/mL DNase I solution (cat# 4536282001, Sigma). After enzymatic dissociation, the cell suspension was passed through a 100 pM cell strainer (cat# 11814389001, Roche) to eliminate macroscopic tissue pieces and then resuspended in 2-volumes of RBC lysis buffer (cat# 11814389001, Roche) for 3 min at room temperature (RT) to eliminate red blood cells. Single cell suspensions were analyzed by flow cytometry.
[0378] Flow cytometry was performed on tumor tissue and PC cell lines (cells at a confluency of 85% were removed from the flask with 1 mM EDTA in PBS). Single-cell suspensions of tumor cells were processed for analysis using PBS with 1% FBS, 2 mmol/L EDTA, and fluorochrome-conjugated mAbs against PSMA (phycoerythrin-conjugated antihuman PSMA, LNI-17, BioLegend #342504). Cells were stained with a live-dead marker to discriminate live cells with Zombie NIR fixable viability dye (BioLegend), according to the manufacturer’s instruction. After incubation, cells were washed and analyzed with a BD LSR Fortessa flow cytometer. Data were analyzed using the Flow Jo software.
[0379] Tumors were harvested and fixed in 10% neutral -buffered formalin (cat# 5701, Thermo Scientific) overnight. Tissues were washed thoroughly under running tap water followed by processing using ethanol, and embedded in paraffin according to standard protocols. Sections (5 pm) were prepared for antibody detection and hematoxylin and eosin staining. Slides were prepared using consecutive sections. For antigen retrieval, slides were incubated for 60 min in citrate buffer at pH 6 at 98 °C. Slides were incubated with a mouse mAb against PSMA (cat# M3620, Dako) at a dilution of 1 :400. PSMA detection was performed using the Refine Detection DAB Kit (Leica). Protein blocking was performed using Protein-Block solution (cat# X0909, DAKO Agilent Technologies) for 10 mins at RT. Mouse cross-reactivity was minimized using a biotinylated anti-mouse antibody (cat# BP-9200, Vector Laboratories). Immunohistochemistry staining for PSMA was evaluated according to the percentage of tumor cells with nuclear positivity shown by Ki-67 staining (cat# RM-9106-R7, clone SP6, Histocom AG) using an Aperio ImageScope (Leica).
Results
[0380] Fluorescence-activated cell sorting (FACS) analysis before tumor cell injection into mice confirmed that 22Rvl cells expressed intermediate and heterogenous levels of PSMA (FIG. 38). Fluorescence minus one (FMO) is represented by the histogram on the left. Geometric mean fluorescence intensity (GMFI) is indicated t by the histogram on the right). Live cells were discriminated by live/dead dye by fluorescent analysis.
[0381] Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 39 and 40, respectively. Data are presented as mean ± SEM or mean ± SD respectively.
[0382] Treatment with ALD101 in the 22RvlCDX model expressing intermediate and heterogeneous levels of PSMA per cells (FIG. 38) caused tumor growth inhibition (TGI) for ~20 days at the two dose levels investigated (10 and 15 mg/kg), reaching a maximum TGI of 45-50% of the initial tumor volume on Day 12. TGI >40% was observed for at least 7 days at both dose levels (FIG. 39).
[0383] Overall, treatment with ALD101 was well tolerated by all 22Rvl CDX mice that received one dose of 10 mg/kg ALD101, as well as by all CDX mice that received one dose of ALD101 15 mg/kg, with no body weight loss (FIG. 40) and no clinical signs of distress observed. No animal deaths were reported during the study.
[0384] PSMA expression was determined in tumor tissues that were collected from castrated CDX mice after treatment with ALD101 (upon tumor regrowth) (FIG. 41). FIG. 41 A shows PSMA expression analyzed by flow cytometry on live cells from tumor tissue collected from CDX mice treated with ALD101 and followed for tumor growth. Tissue was digested to obtain a single cell suspension. Bars show the expression levels of PSMA on tumor tissue after treatment with vehicle (left bar), 10 mg/kg ALDI 01 (middle bar), and 15 mg/kg ALD101 (right bar). Data are shown as mean of geometric mean fluorescence intensity (GMFI) ± SD. Each dot represents a single mouse. FIG 41B shows immunohistochemical staining on tumor tissue for expression of hematoxylin and eosin (HE; upper panel), Ki -67 (middle panel), and PSMA (lower panel) after treatment with vehicle (left panel), 10 mg/kg ALD101 (middle panel), or 15 mg/kg ALD101 (right panel). Scale bar = 300 pm. Treatment with ALD101 at either dose (10 or 15 mg/kg) did not diminish PSMA expression in tumors that had started to regrow following initial inhibition of tumor cell proliferation. EXAMPLE 12
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATIONS (10 MG/KG TWICE) COMPARED TO A SINGLE DOSE OF 10 OR 20 MG/KG OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE
XENOGRAFT MODEL (22Rvl)
[0385] This study was conducted according to the general procedure illustrated in FIG. 42 to evaluate the antitumoral efficacy of repeated dosing of ALD101 at 10 mg/kg administered at 10 days interval compared to a single dose of 10 or 20 mg/kg in a 22Rvl CDX model expressing intermediate and heterogeneous levels of PSMA which differentiates from the high PSMA expressing LNCaP-abl CDX model described above.
[0386] Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. ALD101 solutions were prepared as described in Example 3. Cell lines, cell culture, inoculation and animal experiments were as described in Example 11.
[0387] Mice were castrated ten days before tumor cell injection. A single preparation of 2xl06 22Rvl cells was injected subcutaneously (sc) in the dorsal mouse flank. Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. When tumors reached a volume of approximately 200 mm3 (range -100-350 mm3) roughly 3 weeks after injection, the mice were randomly assigned to a treatment group or vehicle according to their tumor volume to ensure that the tumor volume in different groups was similar and treated as described in Table 23. The date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
Table 23: ADC treatment plan to evaluate antitumor efficacy at a single versus repeated ADC doses in a 22Rvl Model.
Figure imgf000094_0001
*Note that 6 animals were initially treated in this group but 1 animal presenting signs of tumor ulcerations/necrosis was sacrificed and was excluded from grouped analyses.
[0388] After ADC treatment, xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm3 or after 79 days, and animals were sacrificed.
Results
[0389] Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 43 and 44, respectively. Data are presented as mean ± SEM and mean ± SD respectively.
[0390] Major tumor growth inhibition (TGI) was seen in these 22Rvl CDX tumor bearing mice treated with two doses of ALD101 10 mg/kg at 10-day interval, with mean TGI > 90% for 39 days while in mice treated with one dose of ALDI 01 10 or 20 mg/kg, TGI - 90% was only transiently observed (around Day 14).
[0391] Fractionating the single dose of ALD101 20 mg/kg into two administrations of ALD101 10 mg/kg each, administered with a 10-day interval, resulted in TGI lasting more than three times as long (53 days compared with 14 days with a single dose at 20 mg/kg) in this model expressing intermediate and heterogenous levels of PSMA.
[0392] Overall, treatment with ALD101 was well tolerated by all CDX mice that received two doses of 10 mg/kg ALD101 with an interval of 10 days, as well as by all CDX mice that received one dose of ALD101 10 or 20 mg/kg, with no body weight loss and no clinical signs of distress observed. No animal deaths were reported during the study.
[0393] Repeated administrations of low doses of ALD101 (giving transient TGI -50- 80% for a few days as single dose) with a 10-day interval resulted in a substantial increase of TGI > 90% sustained for several weeks compared to an equivalent dose given as a standard single dose in the 22Rvl CDX model. Specifically, in this model expressing intermediate and heterogeneous PSMA levels per cell (see FIG. 38 described in Example 11), repeated dosing achieves complete and sustained TGI response comparable to that obtained in the high PSMA expressing LNCaP-abl CDX model. This finding allows the adjustment and optimization of the dosing regimen according to intermediate or high PSMA levels in patients. EXAMPLE 13
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATION (7.5 MG/KG X2 OR X3 DOSES) OF ALD101 COMPARED TO A SINGLE DOSE OF 7.5 OR 20 MG/KG OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (22Rvl)
[0394] This example was performed according to the general procedure outlined in FIG. 45 to demonstrate the antitumoral efficacy of single or repeated dosing of ALD101 at 7.5 mg/kg administered twice or thrice at 10-day intervals, compared to a single dose of 7.5 or 20 mg/kg (using pooled data from 2 studies) in the 22Rvl CDX model expressing intermediate and heterogeneous levels of PSMA which differentiates this model from the high PSMA expressing LNCaP-abl CDX model described above. Cell lines, cell culture, inoculation and animal experiments were as described in Example 11.
[0395] Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 and prepared according to the general procedure outlined in Example 3.
[0396] Mice were castrated 10 days before tumor cell injection. A single preparation of 2-3 xlO622Rvl cells was injected subcutaneously (sc) in the dorsal mouse flank. Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. When tumors reached a volume of approximately 200 mm3, roughly 3-4 weeks after injection, the mice were randomly assigned to a treatment group or vehicle according to their tumor volume to ensure that the tumor volume in different groups was similar, and treated as described in Table 24. The date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
Table 24: ADC treatment plan to evaluate antitumor efficacy at a single versus repeated
ADC doses in a 22Rvl CDX Model
Figure imgf000096_0001
Figure imgf000097_0001
Note that 38 animals were initially treated but 2 animals presenting signs of tumor ulcerations/necrosis were sacrificed and were excluded from grouped analyses (1 in the ALD101 7.5 mg/kg once and 1 in the ALD101 7.5 mg/kg twice), thus 36 animals were included in the analysis.
[0397] After ADC treatment, xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm3 or after 70 days, and animals were sacrificed.
Results
[0398] Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 46 and 47, respectively. Data are presented as mean ± SEM or mean ± SD respectively.
[0399] Major tumor inhibition was seen in tumor bearing mice treated with three doses of ALD101 7.5 mg/kg with TGI > 90% for 42 days (up to day 59). In tumor bearing mice treated with a single dose at 20 mg/kg or two doses of ALD101 7.5 mg/kg, 90% TGI was observed up to day 14 and 21 respectively. TGI (up to 70%) was transient, lasting for 7 days with a single dose at 7.5 mg/kg. Administering two or three doses of 7.5 mg/kg with a 10-day interval, demonstrated TGI lasting at least 21 and 59 days, respectively.
[0400] Fractionating the single dose of ALD101 20 mg/kg into three administrations of ALD101 7.5 mg/kg each, administered with 10-day intervals, resulted in TGI lasting four times longer (59 days compared to 14 days with a single dose at 20 mg/kg).
[0401] Overall, treatment with ALD101 was well tolerated by all CDX mice that received two or three doses of 7.5 mg/kg ALD101 with an interval of 10 days, as well as by all CDX mice that received one dose of ALD101 7.5 or 20 mg/kg, with no body weight loss and no clinical signs of distress observed. No animal deaths were reported during the study. EXAMPLE 14
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATION (5 MG/KG X2 OR X3 DOSES) OF ALD101 COMPARED TO A SINGLE DOSE OF 5 OR 15 MG/KG OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (22Rvl)
[0402] This example was performed according to the general procedure outlined in FIG. 48 to demonstrate the antitumoral efficacy of single or repeated dosing of ALD101 at 5 mg/kg administered twice or thrice at 10-day intervals, compared to a single dose of 5 or 15 mg/kg in the 22Rvl CDX model expressing intermediate and heterogeneous levels of PSMA which differentiates this model from the high PSMA expressing LNCaP-abl CDX model described above. Cell lines, cell culture, inoculation and animal experiments were as described in Example 11.
[0403] Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 and prepared according to the general procedure outlined in Example 3.
[0404] Mice were castrated 10 days before tumor cell injection. A single preparation of 3 xlO622Rvl cells was injected subcutaneously (s.c.) in the dorsal mouse flank. Xenograft monitoring started 1 week after PC cell injection and was performed three times per week. When tumors reached a volume of approximately 200 mm3, 3 weeks after injection, the mice were randomly assigned to a treatment group or vehicle according to their tumor volume to ensure that the tumor volume in different groups was similar, and treated as described in Table 25. The date of randomization was denoted as Day 0 (DO) and corresponded to the treatment administration day per study design.
Table 25: ADC treatment plan to evaluate antitumor efficacy at a single versus repeated
ADC doses in a 22Rvl CDX Model
Figure imgf000098_0001
Figure imgf000099_0001
[0405] After ADC treatment, xenograft growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of -1500 mm3 or after 39 days, and animals were sacrificed.
Results
[0406] Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 49 and 50, respectively. Data are presented as mean ± SEM or mean ± SD respectively.
[0407] Major tumor inhibition was seen in tumor bearing mice treated with three doses of ALD101 5 mg/kg with TGI > 80% for 16 days (up to day 29). In tumor bearing mice treated with a single dose at 15 mg/kg or two doses of ALD101 5 mg/kg, 80% TGI was observed up to day 22. TGI was transient, lasting for 8 days with a single dose at 5 mg/kg. Administering two or three doses of 5 mg/kg with a 10-day interval, demonstrated TGI lasting at least 22 and 29 days, respectively.
[0408] Fractionating the single dose of ALD101 15 mg/kg into three administrations of ALD101 5 mg/kg each, administered with 10-day intervals, resulted in TGI lasting 1.3 times as long (29 days compared to 22 days with a single dose at 15 mg/kg).
[0409] Overall, treatment with ALD101 was well tolerated by all CDX mice that received two or three doses of 5 mg/kg ALD101 with an interval of 10 days, as well as by all CDX mice that received one dose of ALD101 5 or 15 mg/kg, with no body weight loss and no clinical signs of distress observed. No animal deaths were reported during the study.
EXAMPLE 15
ANTITUMOR ACTIVITY OF REPEATED ADMINISTRATIONS (10 MG/KG TWICE) COMPARED TO SINGLE ADMINISTRATION (10 OR 20 MG/KG ONCE) OF ALD101 IN A PATIENT-DERIVED XENOGRAFT MODEL OF CASTRATION-RESISTANT PROSTATE CANCER (C5)
[0410] This example was performed according to the general procedure illustrated in FIG. 51 to evaluate the antitumoral activity of a single or repeated doses of ALD101 in a C5 patient-derived xenograft (PDX) immunodeficient NRG (NOD-Raglnull IL2rgnull) mouse model of CRPC. This study was conducted to demonstrate the anti turn oral efficacy of repeated dosing of ALD101 at 10 mg/kg administered at a 10-day interval compared to a single dose of 10 mg/kg or 20 mg/kg in this PDX C5 model expressing high levels of PSMA.
[0411] Antibody drug conjugate ALD101 was diluted in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. ALD101 solutions were prepared as described in Example 3.
[0412] Patient-derived cells were a gift from Dr. Tobias Lange (Institute of Anatomy and Experimental Morphology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany). Briefly, tumor tissue was collected at the time of surgery and processed under sterile conditions. Small fragments of the tumor were implanted subcutaneously into the flank of immunocompromised NRG mice. Once tumors were established, C5 cells were serially transplanted from one generation of mice to another to maintain the tumor model.
[0413] Single cell suspensions of C5 cells were prepared for tumor inoculation for mouse model development, with determination of PSMA expression in C5 cells prior to inoculation as analyzed by flow cytometry. PSMA expression was also determined in tumor tissue from PDX mice that were evaluated for ADC antitumor activity. Tumors were harvested and digested to create a single cell suspension.
[0414] Tumor tissue was isolated, minced and processed. Briefly, chopped tumor was digested in 2 ml of Digestion Buffer composed of RPMI supplemented with 200 U/mL of collagenase D from Clostridium histolyticum (cat# 11-088858001, Roche) and 50 U/mL DNase I solution (cat# 4536282001, Sigma) for 30 min to 1 hour at 37 °C on a shaker. After enzymatic dissociation, the cell suspension was passed through a 100 pm cell strainer (cat# 11814389001, Roche) to eliminate macroscopic tissue pieces and then resuspended in 2-volumes of RBC Lysis Buffer (cat# 11814389001, Roche) for 3 min at room temperature to eliminate red blood cells.
[0415] Flow cytometry was performed on tumor tissue harvested from PDX mice prior to inoculation and also after ADC treatment (after tumor regrowth), as described in Example 11. Immunohistochemical staining on tumor tissue of hematoxylin and eosin and PSMA after treatment with vehicle or ADC (after tumor regrowth) was performed as described in Example 11.
[0416] All animal experiments were carried out as described in Example 11. All in vivo studies used 6-8-week-old male immunodeficient NOD-Raglnull IL2rgnull (NRG) mice. Mice were inoculated with 4xl06 PDX C5 cells. Cells were injected subcutaneously into the mouse flank using 1 : 1 Matrigel® Matrix (cat# 354234, Corning) with PBS. The single cell suspension was kept on ice until injection. Monitoring and data collection was performed as described in Example 11.
[0417] Randomization was performed for antitumor evaluation when the mean tumor volume reached approximately 200 mm3 (range -150-300 mm3). The mice were randomized according to their tumor volume to ensure that the tumor volume in different groups was similar (Table 26). The day of randomization and treatment was denoted as Day 0 (DO).
Table 26: ADC treatment plan to evaluate antitumor efficacy of a single ADC dose in a C5 PDX model
Figure imgf000101_0001
[0418] After randomization, mice were treated with a single intravenous dose of ALD101 or vehicle (Table 26). Tumor growth was monitored at 24, 48, and 72 hours together with mice body weight. After this initial daily observation, CDX tumor growth was monitored 3 times per week until the tumor reached a volume of - 1500 mm3 or after 54 days, and animals were sacrificed. Tumor tissue was then collected from each mouse for formalin-fixed paraffin- embedded (FFPE) blocks for immunohistochemistry, snap-frozen tissue samples, and for single cell suspension flow cytometry. The antitumor activity was expressed as percentage of tumor growth inhibition (%TGI) which is the percentage of mean tumor reduction compared to mean initial tumor volume.
Results
[0419] FACS analysis before tumor cell injection into mice confirmed that C5 cells expressed high levels of PSMA (FIG. 52). Histograms represent the geometric mean fluorescence intensity (GMFI) in the C5 cell line (right histogram) and in the control (FMO; left histogram). GMFI represents levels of PSMA expression. Cells were gated on live cells. Live cells were discriminated by live/dead dye by fluorescent analysis.
[0420] Tumor growth curves and mouse body weight for each treatment group from treatment start until study termination are shown in FIGS. 53 and 54, respectively. Data are presented as mean ± SEM or mean ± SD respectively. [0421] ALD101 showed antitumor effects in vivo in the PDX C5 immunodeficient NRG non-castrated mouse model with a mean tumor volume of 200 mm3 at the time of treatment. Major tumor inhibition was seen in all mice treated with two doses of ALD101 10 mg/kg, with mean tumor growth inhibition (TGI) > 90% for 12 days, as well as in mice treated with a single dose of ALD101 20 mg/kg, with mean TGI > 90% for 5 days. TGI was durable lasting for 33 days at 10 mg/kg (two doses) and 28 days at 20 mg/kg with single dose administration (FIG. 53).
[0422] Fractionating the single dose of ALD101 20 mg/kg into two administrations of ALD101 10 mg/kg each, administered with a 10-day interval, resulted in -20% longer TGI (for 33 days) compared with 28 days with a single dose at 20 mg/kg (FIG. 53) in this PDX C5 model.
[0423] Overall, treatment with ALD101 was well tolerated by all PDX C5 mice with no body weight loss (FIG. 54) and no clinical signs of distress observed. No animal deaths were reported during the study.
[0424] Tumor PSMA expression was maintained after ALD101 administration in tumors that had started to regrow following initial inhibition of tumor cell proliferation (FIG. 55). FIG. 55A: PSMA expression was analyzed by flow cytometry on live cells from tumors collected from PDX C5 mice treated with ALD101 and followed for tumor growth for 24-54 days. Tissue was digested to obtain a single cell suspension. Bars show the expression levels of PSMA on tumor tissue after treatment with vehicle (black bar), 10 mg/kg ALD101 (grey bar), 10 mg/kg ALD101 twice at DO and D10 (hashed grey) and 20 mg/kg (white bar). Data are shown as mean of geometric mean fluorescence intensity (GMFI) ± SD representing levels of PSMA expression. Each dot represents a single mouse. FIG. 55B: Immunohistochemical staining on tumor tissue for expression of hematoxylin and eosin (HE; upper panel) and PSMA (lower panel) after treatment with vehicle for 24 days or with ALD101 for 52-54 days with 10 mg/kg once, 10 mg/kg twice or 20 mg/kg once. Scale bar = 200 pm.
EXAMPLE 16
INTERNALIZATION OF ALD101 IN PROSTATE CANCER CELL LINES
[0425] This example evaluates the internalization properties of ALD101 in LNCaP-abl and 22Rvl prostate cancer (PC) cell lines exposed to ALD101, using fluorescence-activated cell sorting (FACS) and immunofluorescence. [0426] Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. ADC-Control (ADC-ctrl) was formulated in 20 mM histidine, pH5.5. The ADC-Control was a human IgGl Kappa isotype CTL antibody that did not bind to PSMA conjugated to LD038. Formulations were prepared in the biosafety cabinets. ADCs were thawed on ice and kept on ice until use to minimize the formation of ADC aggregates. ADC stock solutions were resuspended gently and diluted in cell culture medium (appropriate for the cell line) to prepare the dosing solutions.
[0427] The LNCaP-abl human PC cell line was a gift from Prof. Myles Brown (Dana- Farber Cancer Institute, Boston). The LNCaP-abl cell line was cultured in phenol red-free-RPMI 1640 (cat# 11835063, Life Technologies) supplemented with 10% charcoal- stripped serum (CSS, PAN-Biotech GmbH, Aidenbach, Germany) and 1% penicillin-streptomycin.
[0428] The 22Rvl cell line was purchased from American Tissue Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium with L-glutamine (cat# 21875-034, Life Technologies) supplemented with 10% fetal bovine serum (FBS-11A, Capricorn Scientific) and 1% penicillin/ streptomycin (cat# 15140-122, Life Technologies).
[0429] All cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. All cell lines were periodically checked for Mycoplasma spp. infection using the MycoAlert™ kit (Lonza), according to the manufacturer’s instructions.
[0430] LNCaP-abl and 22Rvl cells were seeded at 1.5xl06 cells/well in a 6 well plate and grown until ~ 85% confluence. ALD101 or ADC-ctrl were conjugated with fluorochrome- conjugated secondary antibodies by incubating each ADC for 30 minutes at room temperature (RT) with an anti-human IgG Fc fragment specific conjugated with Alexa Fluor® 647-labelled AffiniPure goat anti-human IgG, Fc fragment specific (cat# 109-605-098, Jackson ImmunoResearch) diluted 1 : 100 in 1% BSA in PBS.
[0431] Cells were incubated with the dye-conjugated ADC-AF647 or the ADC-ctrl- AF647 at 4°C for 1 hour at 0.1 nM to allow for ADC binding while preventing its internalization. This concentration corresponds to ALD101 IC50 calculated in vitro by sulforhodamine B (SRB) cytotoxicity assay as described in Example 3. After incubation at 4°C, the supernatant was carefully removed, and fresh medium was added. For the TO timepoint cells were immediately harvested. For the other timepoints cells were incubated at 37°C for 1 hour or 3 hours to allow for internalization. At the indicated time points cells were harvested, washed with 1% BSA in PBS and then analyzed by flow cytometry (BD Fortessa). [0432] LNCaP-abl and 22Rvl cells were seeded at 2.5xl04 cells/well in a p-slide 8-well ibitreat (#80826, IBIDI GmbH) and grown until ~ 85% confluence. Cells were incubated with ALD101 for 1 hour at 4°C to allow for ADC binding while preventing its internalization. After incubation, cells were either immediately fixed (for TO timepoint) with 4% (w/v) formaldehyde at RT for 20 minutes or incubated for the indicated time points (0.5, 1, 3, or 6 hours) in the incubator at 37°C and then fixed. After fixation, cells were incubated with glycine-based blocking solution (0.1M in 1% of PBS) for 10 min at RT. Cells were washed three times with 3% BSA in lx PBS for 10 min at RT. Cells were then permeabilized and stained for 30 min at RT by using permeabilization solution (0.05% saponin, 1% BSA in 1 x PBS) containing 1 : 100 of secondary antibody reagent anti-human IgG Fc fragment specific conjugated with Alexa fluor 647 (cat# 109-605-098, Jackson ImmunoResearch Laboratories) and Alexa Fluor 488 conjugated anti-human LAMPl/CD107a (#IC7985G; Bio-Techne AG) or phalloidin (Alexa Fluor plus 555; #A30106, Thermo Fisher Scientific) before mounting the coverglass. Cells were washed three times with 3% BSA in PBS and once with PBS. Stained slides were mounted with coverglasses using the VECTASHIELD antifade mounting medium containing DAPI (#VC-H-1200, Adipogen AG). Photos were acquired with a Leica Stellaris 5 microscope.
Results
[0433] Internalization by immunofluorescence and FACS are shown in FIGS. 56A and 56C, and 56B and 56D, respectively. Colocalization with lysosomes by immunofluorescence is shown in FIGS. 57A-57B. Internalization of ALD101 bound to the cell surface was seen as early as 1 hour after treatment (FIG. 56). ALD101 colocalized with lysosomes as soon as 1 hour after binding (FIG. 57). ALD101 internalization was evident in both the LNCaP-abl cell line, which shows high and homogeneous PSMA expression (FIGS. 56A-56B and 57A), and the 22Rvl cell line which has intermediate and heterogenous PSMA expression (FIGS. 56C-56D and FIG. 57B).
[0434] Maximum binding of ALD101 (high geometric fluorescence intensity [GMFI] and histogram shift to the right relative to fluorescence minus one [FMO]; see FIGS. 56B and 56D) was seen at TO. After 1 hour the signal decreased due to internalization of ALD101 into cells. Full internalization occurred within 3 hours as shown by FACS (FIGS. 56B and 56D). EXAMPLE 17
PHARMACOKINETICS OF SINGLE ADMINISTRATION OF ALD101 (10 MG/KG) IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0435] This study was conducted according to the general procedure illustrated in FIG. 58 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan), total IgG, and total ADC in mouse tumor tissue and plasma after the administration of ALD101 10 mg/kg in an LNCaP-abl CDX castrated immunodeficient mouse model, either NRG (NOD- Raglnull IL2rgnull) or NSG (NOD.Cg-PrkdcSCID I12rgtmlWjl/SzJ) using pooled data from 3 studies.
[0436] Total antibody is defined as the totality of anti-PSMA antibodies that are either unconjugated (for example, not conjugated to any payload molecules) or conjugated to at least one payload molecule. Total ADC is defined as anti-PSMA antibodies conjugated to one or several payloads.
[0437] The method for analyzing free exatecan, based on a published method (Oguma et al, 2005, Chromatogr B Analyt Technol Biomed Life Sci 818:249-256), has been developed and validated previously, and the methods are incorporated herein by reference.
[0438] The method for analyzing total IgG and total ADC in plasma has been developed and validated, the methods for which are described below. Total IgG and total ADC were measured by ligand-binding immuno-affinity capture followed by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). A signature peptide (ASQDVGTAVDWYQQKPGPSPK (SEQ ID NO: 16)) that is unique to ALD101 was used as a surrogate analyte in the total IgG method. Exatecan released after enzymatic linker digestion was used as a surrogate analyte in the total ADC method.
[0439] Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 or in 20 mM histidine, pH 5.5. See Example 3 and Example 10 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions. In each study, 3xl06 LNCaP-abl cells were inoculated per mouse, and randomization was performed when mean tumor volume was -400 or 650 mm3.
[0440] Xenograft monitoring started 1 week after injection of LNCaP-abl PC cells and tumor volumes were measured 3 times per week. As soon as tumors reached a volume of 400, 480 or 650 mm3 (approximately 2-3 weeks after injection), the mice were randomized to ensure comparable tumor volumes in different groups, and treated as described in Table 27. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 27.
Table 27: Experimental plan for PK evaluation after single administration of ALD101 10 mg/kg
Figure imgf000106_0001
[0441] At the sampling times reported in Table 27, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC- MS/MS analysis. Sample Preparation for Quantitative Determinations
(a) Free Exatecan in Mice Plasma and Tumor Tissue
[0442] Prior to analysis, plasma was thawed at room temperature (RT). Tissue samples were weighed (frozen) and 1 gram was homogenized in 9 mL of PBS to obtain a 1 :9 (w/v) homogenate and immediately processed for HPLC-MS/MS analysis.
[0443] Ten microliters of [2Hs]-exatecan (500 ng/mL) were added as an internal standard (IS) to 100 pL of plasma or to 200 pL of tumor or liver homogenate prepared 1 :9 (w/v) in PBS. Samples were vortexed and 500 pL methanol and 0.1% formic acid was added to precipitate proteins. Samples were centrifuged at 15000 ref for 10 min at 4°C. The supernatants were transferred into clean tubes and evaporated under gentle N2 flux at 37 °C. The residues were reconstituted in 200 pL of a solution of (9: 1 ratio of [water + 0.1% formic acid: methanol + 0.1% formic acid]), then vortexed and centrifuged at 15000 ref for 10 min at 4 °C. Supernatants were transferred into an autosampler glass vial and a 5 pL sample was injected into the HPLC-MS/MS system (see Calibration Curve and Quality Control Section - see Example 4).
(b) Total IgG in Mice Plasma
[0444] Prior to analysis, plasma was thawed at RT and immediately processed for LC- MS/MS analysis. Fifty microliters of Dynabeads M-280 with immobilized anti-human IgG (Fc) were added to 50 pL of plasma. Samples were supplemented with 100 pL of PBS and then incubated for immunocapture, gently mixed for 1 hour at RT. Beads were separated with a magnet and supernatant was discarded. Beads were washed twice with PBS-Tween 0.01% and twice with PBS. Urea 8 M in NH4HCO3 100 mM was added followed by DTT 100 mM in NH4HCO3 25 mM for reduction (30 minutes at 37 °C). IAA 200 mM in NH4HCO3 25 mM was added for alkylation (30 minutes RT in the dark). The ADC was digested with Trypsin/Lys-C for 3 hours at 37 °C, and 450 pL of NH4HCO3 100 mM were then added to reduce urea concentration. Digestion was continued overnight at 37 °C. Digestion was quenched adding HCOOH 10%. Ten microliters of ASQD-SIL 500 ng/mL were added as IS. Beads were separated on a magnet and supernatant was collected and dried in a vacuum concentrator. Samples were reconstituted with 100 pL TFA 0.5% and then cleaned and desalted with Cl 8 tips. Samples were dried again in a vacuum concentrator and finally the residues were reconstituted in 100 pL of a solution of (9.5:0.5 ratio of [water + 0.1% formic acid:acetonitrile + 0.1% formic acid]), then vortexed and centrifuged at 15000 ref for 10 minutes at 4 °C. Supernatants were transferred into an autosampler glass vial and a 10 pL sample was injected into the HPLC- MS/MS system.
(c) Total ADC in Mice Plasma
[0445] Prior to analysis, plasma was thawed at RT and immediately processed for LC- MS/MS analysis. Fifty microliters of Dynabeads M-280 with immobilized anti-human IgG (Fc) were added to 50 pL of plasma. Samples were supplemented with 100 pL of PBS and then incubated for immunocapture, gently mixing for 1 hour at RT. Beads were separated with a magnet and supernatant was discarded. Beads were washed twice with PBS-Tween 0.01% and twice with PBS. Sodium acetate 100 mM pH 5.0 was added followed by 15 pL of activated cathepsin-B (25 ng/mL). Linker digestion was performed overnight at 37 °C. The reaction was quenched by TFA 20%. Ten microliters of [2Hs]-exatecan (100 ng/mL) were added as an IS. Samples were vortexed and 500 pL methanol and 0.1% formic acid was added to precipitate proteins. Samples were centrifuged at 15000 ref for 10 minutes at 4°C. The supernatants were transferred into clean tubes and evaporated under gentle N2 flux at 37 °C. The residues were reconstituted in 200 pL of a solution of (9: 1 ratio of [water + 0.1% formic acid:methanol + 0.1% formic acid]), then vortexed and centrifuged at 15000 ref for 10 minutes at 4 °C. Supernatants were transferred into an autosampler glass vial and a 5 pL sample was injected into the HPLC- MS/MS system.
[0446] The calibration curve for free exatecan was built with a blank plasma or homogenized tissue sample from mice, a zero-blank plasma or tissue, 1 sample at the lower limit of quantification concentration level (LLOQ), followed by 6 control plasma or tissue samples spiked with 10 pL of different exatecan working solutions to obtain the following final concentrations: 0.01, 0.02, 0.075, 0.1, 1, 5, and 10 ng exatecan/sample
[0447] Quality controls (QCs) were prepared by spiking control plasma and homogenized tissue samples with 10 pL of different exatecan working solutions to obtain the following final concentrations: 0.05, 2.5, and 7.5 ng exatecan/sample.
[0448] The calibration curve for total IgG was built with a blank plasma from mice, a zero-blank plasma, 1 sample at the LLOQ, followed by 4 control plasma samples spiked with 5 pL of different ALD101 working solutions to obtain the following final concentrations: 0.5, 1, 5, 10, and 50 pg ALDlOl/mL.
[0449] QCs were prepared by spiking control plasma samples with 5 pL of ALD101 working solutions to obtain the following final concentration: 6 pg ALDlOl/mL. [0450] The calibration curve for total ADC was built with a blank plasma or homogenized tissue sample from mice, a zero-blank plasma or tissue, 1 sample at the LLOQ, followed by 5 control plasma samples spiked with 5 pL of different ALD101 working solutions to obtain the following final concentrations: 0.1, 0.5, 1, 5, 10, and 50 pg ALDlOl/mL.
[0451] QCs were prepared by spiking control plasma samples with 5 pL of ALD101 working solution to obtain the following final concentrations: 3 pg ALDlOl/mL.
[0452] Validated HPLC-MS/MS methods were used to determine exatecan, total ADC and total IgG concentrations. Analyte detection and quantification for free exatecan and total ADC and total IgG was carried out by MS/MS using a TSQ Altis spectrometer.
Results
[0453] The biodistribution of the free payload exatecan was analyzed in tumor and plasma from 5 minutes to 10 days after single dose administration of ALDI 01 10 mg/kg (FIG 59). The level of exatecan in the systemic circulation did not reach more than 0.8 ng/mL at the 5- minute timepoint before decreasing to levels below 0.5 ng/mL at 6 hours after 10 mg/kg ALD101 administration. The level of free exatecan in plasma was below LLOQ (0.1 ng/mL) 10 days after ALD101 administration (10 mg/kg).
[0454] Free exatecan was detected in tumor tissues 5 min after treatment administration. Mean tumor concentrations were approximately 80-fold higher than in plasma 2 hours after treatment and were at least 100-fold higher from 6 hours up to 6 days after treatment, favoring an excellent therapeutic index.
[0455] The concentration of total IgG (antibody conjugated or not to exatecan payload) and total ADC (antibody conjugated to at least one payload) in plasma were similar and remained high for up to 6 days post-treatment (FIG. 60), decreasing gradually from 24 hours onwards. This allowed high exposure of targeted tumor tissue to the study drug up to 6 days post-administration in vivo.
EXAMPLE 18
PHARMACOKINETICS OF FREE EXATECAN AFTER A SINGLE DOSE OF ALD101 (3, 5, 7.5, 10, 20 MG/KG) IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0456] This study was conducted according to the general procedure illustrated in FIG. 61 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma after one administration of ALD101 in an LNCaP-abl CDX castrated immunodeficient NRG or NSG mouse model. Specifically, this example illustrates the proportionality of free exatecan in plasma and tumor to the administered dose of ALD101 (for doses ranging from 3 to 20 mg/kg) using pooled data from 7 studies.
[0457] The method for analyzing free exatecan was as described in Example 17. Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 or in 20 mM histidine, pH 5.5. See Example 3 and Example 10 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
[0458] Seven to fourteen days after castration (depending on the study), a single preparation of either 2xl06 or 3xl06 LNCaP-abl cells were injected subcutaneously in the animal’s right flank. Inoculation parameters for each study were as provided in Table 28. Xenograft monitoring started 1 week after injection of LNCaP-abl PC cells and tumor volumes were measured 3 times per week. Depending on the study, the randomization was performed when the mean tumor volume reached approximately 200, 400, 480, 500 or 650 mm3 (approximately 2-4 weeks after injection). In each experiment the mice were randomized to ensure comparable tumor volumes in the different groups, and treated as described in Table 28. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 28.
Table 28: Experimental plan for PK evaluation after single administration of ALD101 at 3,
5, 7.5, 10, or 20 mg/kg
Figure imgf000111_0001
[0459] Twenty -four hours after ALD101 administration, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC-MS/MS analysis. Samples from vehicle injected mice (saline solution) were used as blank biological matrix for the calibration curve and quality control (QC).
Results
[0460] Free exatecan in mice plasma and tumor were analyzed at 24 hours after ALD1010 administration as described in Example 17 and is presented in FIGS. 62A-62C. This example illustrates the proportionality of free exatecan in plasma and tumor to the administered dose of ALD101 (for doses ranging from 3 to 20 mg/kg) using pooled data from 7 studies.
These results also show that a very high and sustained tumor/plasma ratio (-80-100) is observed across ALD101 treatment doses ranging from 3 to 20 mg/kg, favoring an excellent therapeutic index across a range of dose levels. EXAMPLE 19
PHARMACOKINETICS OF REPEATED ADMINISTRATION (7.5 MG/KGX2 OR X3 DOSES) OF ALD101 COMPARED TO A SINGLE DOSE OF 7.5 MG/KG OF ALD101 IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0461] This study was conducted according to the general procedure illustrated in FIG. 63 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma, as well as for total ADC and total IgG in plasma after one to three administrations of ALD101 7.5 mg/kg 10 days apart, in an LNCaP-abl CDX castrated immunodeficient NRG (NOD-Raglnull IL2rgnull) mouse model. Specifically, this example shows that exatecan does not accumulate in plasma following multiple treatment doses of ALD101 7.5 mg/kg administered 10 days apart.
[0462] The methods for analyzing free exatecan, total ADC and total IgG were as described in Example 17. Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. See Example 3 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
[0463] Ten days after castration a single preparation of 3xl06 LNCaP-abl cells were injected subcutaneously in the animal’s right flank. Xenograft monitoring started 1 week after injection of LNCaP-abl PC cells and tumor volumes were measured 3 times per week. As soon as tumors reached a volume of approximately 200 mm3 (approximately 2 weeks after injection), the mice were randomized to ensure comparable tumor volumes in the different groups, and treated as described in Table 29. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 29. Table 29: Experimental plan for PK evaluation after single or repeated administration of ALD101 7.5 mg/kg in an LNCaP-abl CDX mouse model
Figure imgf000113_0001
[0464] At the sampling times reported in Table 29, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC- MS/MS analysis.
Results
[0465] Free exatecan in mice plasma and tumor as well as total ADC and total IgG in plasma were analyzed at 24 hours after the first ALD1010 dose, 9 days after the 2nd dose, and 24 hours after the 3rd dose and is presented in FIGS. 64A-64C and 65 respectively. The mean concentration of free exatecan in plasma was 0.55 ng/mL 24 hours after the first dose treatment, and was below the limit of quantification (0.1 ng/mL) 9 days after the second dose of treatment, supporting the absence of systemic free payload accumulation after multiple doses of ALD101 at 7.5 mg/kg. Twenty -four hours after the third dose, free exatecan concentration in plasma increased to 0.49 ng/ml, a concentration comparable to that reached 24 hours after the first dose. The free exatecan concentration in tumor tissue was approximately 75x higher in tumor tissue compared to plasma 24 hours after the first dose favoring an excellent therapeutic index (FIGS. 64A-64C). Due to the very limited tumor material remaining following the second and third treatments with ALD101, exatecan tumor concentrations cannot be reported.
[0466] After both the first and third doses (z.e., 24 hours post-treatment), the concentrations of total IgG and total ADC (assuming constant DAR 8) were similar (~75 pg/mL). Nine days after the second dose, the IgG and total ADC concentrations were 3.13 and 1.23 pg/mL respectively, favoring prolonged tumor exposure to ALD101 (FIG. 65).
I l l EXAMPLE 20
PHARMACOKINETICS OF REPEATED ADMINISTRATION (5 MG/KG TWICE OR THRICE) OF ALD101 COMPARED TO A SINGLE DOSE OF ALD101 5 OR 10 MG/KG IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (LNCAP-ABL)
[0467] This study was conducted according to the general procedure illustrated in FIG. 66 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma after one to three administrations of ALD101 5 mg/kg 10 days apart, compared to a single administration at 10 mg/kg in an LNCaP-abl CDX castrated immunodeficient NRG or NSG mouse model. Specifically, this example shows that exatecan does not accumulate in plasma following multiple treatment doses of 5 mg/kg ALD101 administered 10 days apart. This example also illustrates the proportionality of free exatecan in plasma and tumor to the administered dose of ALD101 using pooled data from 4 studies.
[0468] Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. See Example 10 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
[0469] Ten to fourteen days after castration (depending on the study), a single preparation of 3xl06 LNCaP-abl cells was injected subcutaneously in the animal’s right flank. Xenograft monitoring started 1 week after injection of LNCaP-abl PC cells and tumor volumes were measured 3 times per week. Depending on the study, the randomization was performed when the mean tumor volume reached approximately either 400, 480, 500, or 650 mm3 (roughly 2-4 weeks after injection). Inoculation parameters are described in Table 30. In each experiment the mice were randomized to ensure comparable tumor volumes in the different groups and treated as described in Table 31. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 31.
Table 30: Inoculation parameters for LNCaP-abl CDX mice models
Figure imgf000114_0001
Figure imgf000115_0001
Table 31: Experimental plan for PK evaluation after single or repeated administration of
ALD101 5 or 10 mg/kg in an LNCaP-abl CDX mouse model
Figure imgf000115_0002
[0470] At the sampling times reported in Table 31, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC- MS/MS analysis. Results
[0471] Free exatecan in mice plasma and tumor tissue were analyzed at various time points, as described in Example 17 and presented FIGS. 67A-67B, FIGS 68A-68C and FIGS. 69A-69C
[0472] FIG. 66 shows an in vivo experimental plan for PK evaluation of ALD101 5 mg/kg in an LNCaP-abl CDX model. In mice treated with ALD101 5 mg/kg, the maximal concentration of free exatecan in plasma was 0.27 ng/mL 24h after first dose treatment and then decreased to 0.13 ng/mL 4 days after the first dose. Twenty -four hours after second and third doses, free exatecan concentrations were 0.33 ng/mL and 0.29 ng/mL respectively, which is comparable to those reached after the first dose (FIGS. 67A-67B). This supports the absence of systemic free payload accumulation after multiple doses of ALD101 at 5 mg/kg. The free exatecan concentration in tumor tissue after first dose was >100x higher in tumor tissue compared to plasma between 2 hours and 24 hours post first dosing and remained above ~60x up to 96 hours post dosing, thus supporting an excellent therapeutic index (FIGS. 68A-68C). No accumulation of free exatecan in tumor is observed after the second and third administration of ALD101 5 mg/kg at 10-day interval (FIGS. 68A-68C).
[0473] Free exatecan levels measured in tumor and plasma were proportional to the administered doses, while the exatecan Tumor/Plasma ratio remained high (-80-120) and constant in the ALDI 01 dose range tested (FIGS 69A-69C).
EXAMPLE 21
PHARMACOKINETICS OF FREE EXATECAN IN PLASMA AND TUMOR AFTER A SINGLE DOSE OF ALD101 (10 MG/KG) IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL
(22Rvl)
[0474] This study was conducted according to the general procedure illustrated in FIG. 70 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma after a single administration of ALD101 10 mg/kg in a 22Rvl CDX castrated immunodeficient NRG (NOD-Raglnull IL2rgnull) mouse model. Specifically, this example illustrates the biodistribution of free exatecan 24 hours after a single dose of ALD101 10 mg/kg in a CDX model expressing intermediate and heterogeneous levels of PSMA which differentiates from the LNCaP-abl model that expresses high and homogeneous levels of PSMA described above. [0475] Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. See Example 11 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the 22Rvl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
[0476] Seven days after castration, a single preparation of 2xl06 22Rvl cells was injected subcutaneously in the animal’s right flank. Xenograft monitoring started 1 week after injection of 22Rvl PC cells and tumor volumes were measured 3 times per week. As soon as tumors reached a volume of 200 mm3 (approximately 3 weeks after injection), the mice were randomized to ensure comparable tumor volumes in the different groups, and treated as described in Table 32. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 32.
Table 32: Experimental plan for PK evaluation after single administration of ALD101 10 mg/kg in a 22Rvl CDX mouse model
Figure imgf000117_0001
[0477] Twenty -four hours after ALD101 administration, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC-MS/MS analysis.
Results
[0478] Free exatecan in mice plasma and tumor were analyzed as described in Example 17 and presented FIGS. 71A-71C.
[0479] Twenty -four hours after ALD101 10 mg/kg administration the mean concentration of free exatecan in plasma was 0.73 ng/mL. The free exatecan concentration in tumor tissue was approximately 67x higher in tumor tissue compared to plasma (48 ng/g) favoring a high tumor/plasma ratio of - 67 (FIGS. 71A-71C), and thus supporting an excellent therapeutic index in the 22Rvl CDX model that expresses intermediate and heterogeneous levels of PSMA per cells (see FIG. 38 and Example 11) which differentiates this model from the high PSMA expressing LNCaP-abl CDX model (FIG. 11) described in FIG. 59 of Example 17.
EXAMPLE 22
PHARMACOKINETICS OF FREE EXATECAN IN PLASMA AND TUMOR AFTER A SINGLE DOSE OF ALD101 (10 MG/KG) IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (22Rvl)
[0480] This study was conducted according to the general procedure illustrated in FIG. 72 to characterize the pharmacokinetic (PK) profile of ALD101 free exatecan payload in mouse tumor tissue and plasma, as well as total ADC and total IgG in plasma after a single administration of ALD101 10 mg/kg in a 22Rvl CDX castrated immunodeficient NSG (NOD.Cg-PrkdcSCID I12rgtmlWjl/SzJ) mouse model. Specifically, this example illustrates the biodistribution of free exatecan 2 hours to 6 days after a single dose of ALD101 10 mg/kg in a CDX model expressing intermediate and heterogeneous levels of PSMA which differentiates from the LNCaP-abl model that expresses high and homogeneous levels of PSMA described above.
[0481] Total antibody (IgG) is defined as the totality of anti-PSMA antibodies that are either unconjugated (for example, not conjugated to any payload molecules) or conjugated to at least one payload molecule. Total ADC is defined as antibody conjugated to one or several payloads.
[0482] Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. See Example 10 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions. See Example 11 for additional information on the source, maintenance and culture of the 22Rvl cell line.
[0483] Ten days after castration a single preparation of 2.5xl06 22Rvl cells was injected subcutaneously in the animal’s right flank. Xenograft monitoring started 1 week after injection of 22Rvl PC cells and tumor volumes were measured 3 times per week. As soon as tumors reached a volume of 250 mm3 (approximately 4 weeks after injection), the mice were randomized to ensure comparable tumor volumes in the different groups, and treated as described in Table 33. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 33.
Table 33: Experimental plan for PK evaluation after single administration of ALD101 10 mg/kg in a 22Rvl CDX mouse model
Figure imgf000119_0001
[0484] At the sampling time indicated in Table 33, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC- MS/MS analysis.
Results
[0485] Free exatecan in mice plasma and tumor were analyzed as described in Example 17 and presented FIGS. 73A-73C. Total IgG and total ADC in plasma were analyzed as described in Example 17 and presented FIG. 74.
[0486] Mean exatecan concentrations in mice treated with ALD101 10 mg/kg were high in tumor tissue (73 ng/g), while mean plasma concentrations of the free payload were very low (below 0.46 ng/mL), resulting in a high tumor/plasma ratio, with mean tumor concentrations 70- fold higher than in plasma 2 hours after ALD101 administration and more than 100 fold higher from 6 hours up to 6 days post-treatment (FIGS. 73A-73C), reflecting an enrichment of the drug concentration in tumor tissue in the 22Rvl CDX model expressing intermediate and heterogeneous levels of PSMA per cells (see FIG. 38 from Example 11) which differentiates from the LNCaP-abl CDX model that expresses high PSMA levels. This high tumor/plasma ratio favors an excellent therapeutic index (FIGS. 73A-73C). In mice treated with ALD101 10 mg/kg, the concentration of total IgG (antibody conjugated or not to exatecan payload) and total ADC (antibody conjugated to at least one payload) in plasma were similar and remained high for up to 6 days post-treatment (FIG. 74), decreasing gradually from 24 hours onwards. This allowed high exposure of targeted tumor tissue to the study drug up to 6 days post-administration in vivo.
EXAMPLE 23
PHARMACOKINETICS OF FREE EXATECAN IN PLASMA AND TUMOR AFTER A SINGLE DOSE OF ALD101 (5 OR 7.5 MG/KG) IN A CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODEL (22Rvl)
[0487] This study was conducted according to the general procedure illustrated in FIG. 75 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma after a single administration of ALD101 5 or 7.5 mg/kg in a 22Rvl CDX castrated immunodeficient NRG (NOD-Raglnull IL2rgnull) mouse model. Specifically, this example illustrates the biodistribution and dose proportionality of free exatecan 24 hours after a single dose of ALD101 5 or 7.5 mg/kg in a CDX model expressing intermediate and heterogeneous levels of PSMA which differentiates from the LNCaP-abl model that expresses high and homogeneous levels of PSMA described above.
[0488] Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. An isotype control ADC (IgGl) that does not bind PSMA was used as a control. The CTRL ADC was formulated in the same buffer. See Example 11 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the 22Rvl cell line, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
[0489] Ten days after castration, a single preparation of 3 xlO6 22Rvl cells was injected subcutaneously in the animal’s right flank. Xenograft monitoring started 1 week after injection of 22Rvl PC cells and tumor volumes were measured 3 times per week. As soon as tumors reached a volume of 200 mm3 (roughly 4 weeks after injection), the mice were randomized to ensure comparable tumor volumes in the different groups, and treated as described in Table 34. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 34.
Table 34: Experimental plan for PK evaluation after single administration of ALD101 or CTRL ADC 5 or 7.5 mg/kg in a 22Rvl CDX mouse model
Figure imgf000121_0001
[0490] Twenty -four hours after ALD101 administration, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC-MS/MS analysis.
Results
[0491] Free exatecan in mice plasma and tumor were analyzed as described in Example 17 and presented FIGS. 76A-76C.
[0492] Twenty -four hours after ALD101 5 and 7.5 mg/kg administration the mean concentration of free exatecan in plasma was 0.41 and 0.65 ng/mL respectively, demonstrating low systemic exposure and free exatecan dose proportionality. The free exatecan concentration was approximately 63x higher in tumor tissue compared to plasma at both dose levels and was also proportional to the administered dose (25.6 and 35.4 ng/g respectively) (FIGS. 76A-76C). When administering the non-binding control ADC (ADC CTRL), the drug concentration in tumor and the tumor/plasma ratio were 3-4-fold lower than with ALD101 demonstrating preferential targeting of ALD101 to the tumor. The high tumor/plasma ratio of - 63 for ALD101 at both dose levels, supports an excellent therapeutic index in the 22Rvl CDX model that expresses intermediate and heterogeneous levels of PSMA per cells (see FIG. 38 and Example 11) which differentiates this model from the high PSMA expressing LNCaP-abl CDX model (FIG. 11) described in FIG. 59 of Example 17.
EXAMPLE 24
PHARMACOKINETICS OF FREE EXATECAN AFTER A SINGLE DOSE OF ALD101 (3, 5, 7.5, 10, 20 MG/KG) IN TWO CASTRATED PROSTATE CANCER MOUSE XENOGRAFT MODELS (LNCAP-ABL
AND 22RV1)
[0493] This study was conducted according to the general procedure illustrated in FIG. 77 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma after one administration of ALD101 in two different CDX castrated immunodeficient NRG / NSG mouse models (LNCaP-abl and 22Rvl). Specifically, this example illustrates the proportionality of free exatecan in plasma and tumor to the administered dose of ALD101 (for doses ranging from 3 to 20 mg/kg) and compares the pharmacokinetics of free exatecan in two CDX models expressing different levels of PSMA, the LNCaP-abl CDX model expressing high and homogeneous levels of PSMA while the 22Rvl expresses intermediate and heterogeneous PSMA levels (using pooled data from 10 studies).
[0494] The method for analyzing free exatecan was as described in Example 17 Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5 or in 20 mM histidine, pH 5.5. See Example 3 and Example 10 and 11 for additional information on the description of the ADC used (ALD101), ADC administration, manipulation, the source, maintenance and culture of the LNCaP-abl and 22Rvl cell lines, animal handling procedures (including protocol ethics committee approval), mice castration, tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
[0495] Seven to fourteen days after castration (depending on the study), a single preparation of either 2xl06, 2.5 xlO6 or 3xl06 LNCaP-abl or 22Rvl cells were injected subcutaneously in the animal’s right flank. Inoculation parameters for each study were as provided in Table 35. Xenograft monitoring started 1 week after injection of LNCaP-abl / 22Rvl PC cells and tumor volumes were measured 3 times per week. Depending on the study, the randomization was performed when the mean tumor volume reached approximately 200, 250, 400, 480, 500 or 650 mm3 (approximately 2-4 weeks after injection). In each experiment the mice were randomized to ensure comparable tumor volumes in the different groups, and treated as described in Table 35. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 35.
Table 35: Experimental plan for PK evaluation after single administration of ALD101 at 3, 5,
7,5, 10, or 20 mg/kg in LNCaP-abl or 22Ryl CDX models
Figure imgf000123_0001
[0496] Twenty -four hours after ALD101 administration, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC-MS/MS analysis. Samples from vehicle injected mice (saline solution) were used as blank biological matrix for the calibration curve and quality control (QC).
Results
[0497] Free exatecan in mice plasma and tumor were analyzed at 24 hours after ALD1010 administration in LNCaP-abl and 22Rvl CDX models as described in Example 17 and is presented in FIGS. 78A-78C. This example illustrates the proportionality of free exatecan in plasma and tumor to the administered dose of ALD101 (for doses ranging from 3 to 20 mg/kg) using pooled data from 10 studies. These results also show that a very high and sustained tumor/plasma ratio (-60-120) is observed at 24 hours post administration across ALD101 treatment doses ranging from 3 to 20 mg/kg, thus supporting an excellent therapeutic index. The levels of free exatecan in both plasma and tumor are similar between the LNCaP-abl and 22Rvl CDX models which is remarkable considering LNCaP-abl expresses higher levels of PSMA than the 22Rvl CDX model (intermediate and heterogeneous levels of PSMA).
EXAMPLE 25
PHARMACOKINETICS OF FREE EXATECAN IN PLASMA AND TUMOR AFTER A SINGLE DOSE OF ALD101 (10 MG/KG) IN A A PATIENT-DERIVED XENOGRAFT MODEL OF CASTRATION- RESISTANT PROSTATE CANCER (C5)
[0498] This study was conducted according to the general procedure illustrated in FIG. 79 to characterize the pharmacokinetic (PK) profile of ALD101 free payload (exatecan) in mouse tumor tissue and plasma after a single administration of ALD101 10 mg/kg in a patient- derived xenograft mouse model of castration-resistant prostate cancer (C5 model) immunodeficient NRG NOD-Raglnull IL2rgnull. Specifically, this example illustrates the biodistribution of free exatecan 24 hours after a single dose of ALD101 10 mg/kg in a PDX model expressing high and homogeneous levels of PSMA. Many studies have demonstrated that PDX models can preserve the histopathology and genetic landscape of the parental tumor, moreover clonal compositions in PDX models parallel the tumoral genetic heterogeneity (Guenot et al., 2006).
[0499] Antibody drug conjugate ALD101 was formulated in 20 mM histidine buffer, 8% (w/v) sucrose, 0.02% (w/v) PS80, pH 5.5. See Example 11 and Example 17 for additional information on the description of the ADC used (ALD101), ADC administration and manipulation, the source and maintenance of the C5 cell line, animal handling procedures (including protocol ethics committee approval), tumor cell line inoculation, animal monitoring, data collection, randomization, and experimental termination conditions.
[0500] Mice were inoculated with 4xl06 PDX C5 cells. Cells were injected subcutaneously into the mouse flank using 1 : 1 Matrigel® Matrix (cat# 354234, Coming) with PBS. Xenograft monitoring started 1 week after injection of C5 PC cells and tumor volumes were measured 3 times per week. As soon as tumors reached a volume of 200 mm3 (roughly 4 weeks after injection), the mice were randomized to ensure comparable tumor volumes in the different groups, and treated as described in Table 36. The date of randomization was denoted as DO and corresponds to the treatment administration day per study design, while the time of treatment was Time 0. The treatment groups analyzed in this study are presented in Table 36.
Table 36: Experimental plan for PK evaluation after single administration of ALD101 10 mg/kg in a PDX C5 mouse model
Figure imgf000125_0001
* Six mice were initially treated but one mouse was excluded due to a problem during ALD101 injection. Thus 5 mice were included in the analysis.
[0501] Twenty -four hours after ALD101 administration, blood was collected and mice were immediately euthanized by cervical dislocation. Tumor tissue was collected from each mouse and immediately frozen on dry ice. To obtain plasma, blood was centrifuged at 4000 rpm for 10 minutes at 4°C and frozen on dry ice. All frozen samples were stored at -80 °C until HPLC-MS/MS analysis.
Results
[0502] Free exatecan in mice plasma and tumor were analyzed as described in Example 17 and presented FIGS. 80A-80C.
[0503] Twenty -four hours after ALD101 10 mg/kg administration the mean concentration of free exatecan in plasma was 0.71 ng/mL (FIG. 80A). The free exatecan concentration was approximately 80x higher in tumor tissue compared to plasma (58 ng/g, see FIGS. 80B-C) favoring a high tumor/plasma ratio of ~ 80, and thus supporting an excellent therapeutic index in the PDX C5 model that expresses high levels of PSMA (see FIG. 52 and Example 15).
[0504] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including European Application No. 23307425.1 filed December 29, 2023, U.S. Provisional Patent Application No. 63/627,614 filed January 31, 2024, U.S. Provisional Patent Application No. 63/678,439 filed August 1, 2024, and U.S. Provisional Patent Application No. 63/723,462 filed November 21, 2024, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
[0505] These and other changes can be made to the embodiments in light of the abovedetailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. A conjugate having the structure of Formula (I):
Ab-[L-E]n
(I) wherein:
Ab is an anti-prostate specific membrane antigen (PSMA) antibody, or an antigenbinding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:4, a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:5; and the VL comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:8;
E is, at each occurrence, an exatecan payload; and
L is a linker covalently attached at a first end to the exatecan payload and further covalently attached at a second end to the Ab via a sulfur-containing residue of the Ab, wherein the linker has, at each occurrence, independently one of the following structures:
Figure imgf000128_0001
Figure imgf000129_0001
wherein:
* is the covalent attachment to the Ab; is the covalent attachment to the exatecan payload; m is about 0 to about 20; and n is about 1 to about 20.
2. The conjugate of claim 1, having the following structure (II):
Figure imgf000129_0002
3. The conjugate of claim 1 or 2, wherein m is about 10 to about 20.
4. The conjugate of any one of claims 1-3, having the structure of Formula (III):
Figure imgf000130_0001
wherein each L1 independently has one of the following structures:
Figure imgf000130_0002
wherein * is the covalent bond to the Ab and ** is a covalent bond to the carbon marked with an arrow.
The conjugate of claim 4, wherein each
Figure imgf000130_0003
The conjugate of claim 4, wherein each L1 is
Figure imgf000130_0004
HO. V* n i N
II I l-l
7. The conjugate of claim 4, wherein each L1 is O *
8. The conjugate of any one of claims 1-7, wherein the anti-PSMA antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody, or de-immunized antibody.
9. The conjugate of any one of claims 1-8, wherein the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2.
10. The conjugate of any one of claims 1-9, wherein the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2, wherein the amino acid sequences of HCDRs 1-3 and LCDRs 1-3 are unchanged.
11. The conjugate of any one of claims 1-10, wherein the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO:2.
12. The conjugate any one of claims 1-11, wherein the anti-PSMA antibody or antigen-binding fragment thereof comprises a human IgGl constant region.
13. The conjugate of any one of claims 1-12, wherein the anti-PSMA antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.
14. The conjugate of any one of claims 1-13, wherein the point of attachment of the linker to the anti-PSMA antibody or antigen binding fragment thereof is a cysteine residue of the anti-PSMA antibody or antigen binding fragment thereof.
15. The conjugate of claim 14, wherein the point of attachment is a cysteine residue at position 214 of SEQ ID NO: 12, a cysteine residue at position 218 of SEQ ID NO: 11, a cysteine residue at position 224 of SEQ ID NO: 11, and a cysteine residue at position 227 of SEQ ID NO: 11.
16. The conjugate of any one of claims 1-15, wherein n is about 4 to about 16.
17. The conjugate of any one of claims 1-16, wherein n is about 6 to about 8.
18. The conjugate of claim 17, wherein n is 6.
19. The conjugate of claim 17, wherein n is 7.
20. The conjugate of claim 17 wherein n is 8.
21. The conjugate of claim 17 wherein n is 9.
22. The conjugate of claim 17 wherein n is 10.
23. A conjugate formed by reaction of a linker drug with an antibody, or an antigen binding fragment thereof, wherein the linker drug has the following structure:
Figure imgf000132_0001
wherein m is about 0 to about 20; and the antibody, or antigen binding fragment thereof, is an anti-prostate specific membrane antigen (PSMA) antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:3, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:4, a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:5; and the VL comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:8, wherein the reacting comprises forming a covalent bond between the sulfur atom of one or more cysteine residues of the antibody, or antigen binding fragment thereof, and one or more linker drugs.
24. The conjugate of claim 23, wherein m is about 10 to about 20.
25. The conjugate of claim 23 or 24, wherein n is about 4 to about 16.
26. The conjugate of claim 23 or 24, wherein n is about 6 to about 8.
27. The conjugate of claim 23 or 24, wherein n is 6.
28. The conjugate of claim 23 or 24, wherein n is 7.
29. The conjugate of claim 23 or 24, wherein n is 8.
30. The conjugate of claim 23 or 24, wherein n is 9.
31. The conjugate of claim 23 or 24, wherein n is 10.
32. The conjugate of claim 23, wherein the linker drug has the following structure:
Figure imgf000134_0001
33. The conjugate of any one of claims 23-32, wherein the anti-PSMA antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody, or de-immunized antibody.
34. The conjugate of any one of claims 23-33, wherein the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2.
35. The conjugate of any one of claims 23-34, wherein the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 1, and a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:2, wherein the amino acid sequences of HCDRsl-3 and LCDRsl-3 are unchanged.
36. The conjugate of any one of claims 23-35, wherein the anti-PSMA antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO:2.
37. The conjugate any one of claims 23-36, wherein the anti-PSMA antibody or antigen-binding fragment thereof comprises a human IgGl constant region.
38. The conjugate of any one of claims 23-37, wherein the anti-PSMA antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.
39. The conjugate of claim 38, wherein the one or more linker drugs forms a covalent bond with a cysteine residue at position 214 of SEQ ID NO: 12, a cysteine residue at position 218 of SEQ ID NO: 11, a cysteine residue at position 224 of SEQ ID NO: 11, and a cysteine residue at position 227 of SEQ ID NO: 11.
40. The conjugate of any one of claims 1-39, wherein, when the conjugate is administered to a patient having an intermediate or heterogeneous PSMA -expressing cancer, the conjugate provides a tumor to plasma ratio of free exatecan that is within 30% of the tumor to plasma ratio of free exatecan when the conjugate is administered to a patient having a high PSMA -expressing cancer, preferably wherein the tumor to plasma ratio of free exatacan is within 25%, 20%, 15%, 10% or 5%.
41. The conjugate of claim 40, wherein the tumor plasma ratio of free exatecan is higher when the conjugate is administered to a patient having an intermediate or heterogeneous PSMA -expressing cancer compared to the tumor to plasma ratio of free exatecan when the conjugate is administered to a patient having a high PSMA -expressing cancer.
42. A pharmaceutical composition comprising the conjugate of any one of claims 1- 41, and a pharmaceutically acceptable excipient.
43. A method of treating a subject having a PSMA expressing cancer, comprising administering to a subject in need thereof an effective amount of the conjugate of any one of claims 1-41 or the pharmaceutical composition of claim 42.
44. The method of claim 43, wherein the PSMA expressing cancer is prostate cancer, salivary gland cancer, thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer.
45. The method of claim 43 or 44, wherein the subject has prostate cancer.
46. The method of claim 45, wherein the subject has metastatic prostate cancer.
47. The method of claim 46, wherein the subject has metastatic castrate resistant prostate cancer.
48. The method of claim 44, wherein the subject has salivary gland cancer.
49. The method of any one of claims 43-48, wherein the conjugate is administered systemically.
50. The method of any one of claims 43-49, wherein the conjugate is administered intravenously.
51. The method of any one of claims 43-50, wherein the conjugate is administered at a dose of about 0.1 milligrams of the conjugate per kilogram of the subject (mg/kg) to about 20 mg/kg.
52. The method of any one of claims 43-51, wherein the conjugate is administered at a dose of about 3 mg/kg, at a dose of about 5 mg/kg, at a dose of about 7.5 mg/kg, at a dose of about 10 mg/kg, at a dose of about 15 mg/kg, or at a dose of about 20 mg/kg.
53. The method of any one of claims 43-52, wherein the PSMA-expressing cancer expresses an intermediate or heterogeneous level of PSMA.
54. The method of claim 53, wherein the tumor to plasma ratio of free exatecan in the subject is within 30% of the tumor to plasma ratio of free exatecan in a subject having a high PSMA -expressing cancer, preferably wherein the tumor to plasma ratio of free exateacan is within 25%, 20%, 15%, 10% or 5%.
55. The method of any one of claims 43-54, wherein the tumor to plasma ratio is higher in the subject having a PSMA-expressing cancer that expresses an intermediate or heterogeneous level of PSMA as compared to the tumor to plasma ratio in a subject having a PSMA-expressing cancer that expresses high level of PSMA.
56. A method for treating a subject having a PSMA expressing cancer, comprising administering to a subject in need thereof a predetermined amount of the conjugate of any one of claims 1-41, wherein the predetermined amount is administered to the subject in two or more doses within a 30-day period.
57. The method of claim 56, wherein the predetermined amount is administered to the subject in two doses within the 30-day period.
58. The method of claim 56, wherein the predetermined amount is administered to the subject in three doses within the 30-day period.
59. The method of claim 56, wherein the predetermined amount is administered to the subject in more than three doses within the 30-day period.
60. The method of any one of claims 56-59, wherein the two or more doses are administered at a 7 to 15-day interval.
61. The method of any one of claims 56-59, wherein the two or more doses are administered at a 7-day interval, 8-day interval, 9-day interval or 10-day interval.
62. The method of any one of claims 56-60, wherein the two or more doses are administered within a 20-day period.
63. The method of any one of claims 56-60, wherein the predetermined amount is administered in three doses within a 20-day period.
64. The method of any one of claims 56-63, the administering results in an exatecan tumor to plasma ratio in the subject of at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100:1, at least 110: 1, at least 120: 1, at least 130: 1, at least 140: 1 or at least 150: 1.
65. The method of claim 64, wherein the tumor to plasma ratio is sustained for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or up to at least 2 weeks after administration of the conjugate.
66. The method of any one of claims 43-65, wherein a period of mean tumor growth inhibition (TGI) in the subject is extended compared to administering the predetermined amount in a single dose.
67. The method of claim 66, wherein the period of tumor growth inhibition is extended at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, or at least 400%.
68. The method of any one of claims 43-67, wherein the method results in tumor growth inhibition in the subject for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days.
69. The method of claim 68, wherein the tumor growth inhibition is 60%, 70%, 80%, 90% or greater.
70. The method of any one of claims 43-69, wherein the method results in complete inhibition of tumor growth for at least 40 days, at least 45 days, at least 50 days, at least 55 days or at least 60 days.
71. The method of any one of claims 43-70, wherein the subject has a PSMA expressing cancer with a tumor volume of at least 125 mm3, at least 175 mm3, at least 200 mm3, at least 250 mm3, at least 300 mm3, at least 350 mm3, at least 400 mm3, at least 450 mm3, at least 500 mm3, at least 550 mm3 at least 600 mm3 or at least 650 mm3 prior to administering two or more doses.
72. The method of any one of claims 43-71, wherein the subject is a human.
73. The method of any one of claims 51-72, wherein the PSMA expressing cancer is prostate cancer, salivary gland cancer, thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, glioblastoma, breast cancer, lung cancer, gastric cancer, colorectal carcinoma, and pancreatic cancer.
74. The method of claim 73, wherein the subject has prostate cancer.
75. The method of claim 74, wherein the subject has metastatic prostate cancer.
76. The method of claim 74, wherein the subject has metastatic castrate resistant prostate cancer.
77. The method of claim 72, wherein the subject has salivary gland cancer.
78. The method of any one of claims 43-77, wherein the conjugate is administered systemically.
79. The method of any one of claims 43-77, wherein the conjugate is administered intravenously.
80. The method of any one of claims 43-79, wherein the method further comprises administering an additional therapy.
81. The method of claim 80, wherein the additional therapy comprises surgery, a hormone therapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, a molecularly targeted therapeutic agent, thermotherapy, radiation therapy, or a vaccine.
82. The method of claim 81, wherein the hormone therapeutic agent is an antiandrogen therapeutic agent.
83. The method of any one of claims 80-82, wherein the additional therapeutic agent is bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, proxalutamide, cimetidine, or topilutamide.
84. The method of any one of claims 80-83, wherein the additional therapeutic agent is enzalutamide.
85. The method of claim 80, wherein the additional therapeutic agent is directed to a prostate cancer target other than PSMA.
86. The method of claim 85, wherein the additional therapeutic agent targets (siz- transmembrane epithelial antigen of the prostate 1 (STEAP1) or B7H3 (CD276).
87. The method of any one of claims 56-86, wherein the predetermined amount ranges from about 0.1 milligrams of the conjugate per kilogram of the subject (mg/kg) to about 30 mg/kg.
88. The method of any one of claims 56-86, wherein the predetermined amount is about 3 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg or about 30 mg/kg.
89. The method of any one of claims 56-88, wherein the PSMA-expressing cancer expresses an intermediate or heterogeneous level of PSMA.
90. The method of claim 89, wherein the tumor to plasma ratio of free exatecan in the patient is within 30% of the tumor to plasma ratio of free exatecan in a patient having a high PSMA -expressing cancer, preferably wherein the tumor to plasma ratio of free exateacan is within 25%, 20%, 15%, 10% or 5%.
91. The method of claim 90, wherein the tumor to plasma ratio is higher in the subject having a PSMA-expressing cancer that expresses an intermediate or heterogeneous level of PSMA as compared to the tumor to plasma ratio in a subject having a PSMA-expressing cancer that expresses high level of PSMA.
92. A method for treating a PSMA-expressing cancer in a plurality of subjects, the method comprising: determining the PSMA expression level of the cancer in each subject; classifying each subject as having either: a) a low level of PSMA expression; b) an intermediate or heterogeneous level of PSMA expression; c) a high level of PSMA expression; and administering the conjugate of any one of claims 1-41, or the pharmaceutical composition of claim 42, to a subject classified as having an intermediate or heterogeneous, or high level of PSMA expression.
93. The method of claim 92, further comprising determining an appropriate dose of the conjugate based on the subject’s PSMA expression level.
94. The method of claim 92 or 93, wherein the subject is classified as having an intermediate or heterogeneous level of PSMA expression.
95. The method of claim 92 or 93, wherein the subject is classified as having a high level of PSMA expression.
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