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WO2024026340A1 - Polythérapie pour le traitement du cancer de l'ovaire - Google Patents

Polythérapie pour le traitement du cancer de l'ovaire Download PDF

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Publication number
WO2024026340A1
WO2024026340A1 PCT/US2023/071006 US2023071006W WO2024026340A1 WO 2024026340 A1 WO2024026340 A1 WO 2024026340A1 US 2023071006 W US2023071006 W US 2023071006W WO 2024026340 A1 WO2024026340 A1 WO 2024026340A1
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WIPO (PCT)
Prior art keywords
immunoconjugate
seq
administered
amino acid
chimeric protein
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PCT/US2023/071006
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English (en)
Inventor
Arundathy PANDITE
Fatima RANGWALA
Tom LAMPKIN
Taylor Schreiber
George FROMM
Suresh DE SILVA
Michael William METHOD
Eric Henry WESTIN
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Immunogen Switzerland GmbH
Shattuck Labs Inc
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Immunogen Switzerland GmbH
Shattuck Labs Inc
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Publication of WO2024026340A1 publication Critical patent/WO2024026340A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule

Definitions

  • the present disclosure relates to, inter alia, compositions and methods that find use in the treatment of ovarian cancer.
  • the instant application contains a sequence listing, which has been submitted in XML format via EFS-Web.
  • Combination therapies are very common in modern cancer treatment. However, combination therapies are highly unpredictable. For example, combination therapy may not be efficacious even when drug target pairs are validated.
  • EOC epithelial ovarian cancer
  • standard first-line treatment for patients with EOC is cytoreductive surgery and taxane/platinum-based chemotherapy. Unfortunately, most patients relapse and develop resistance to platinum agents. As a consequence, EOC remains the most lethal gynecologic malignancy. Therefore, new treatment options are needed to significantly increase survival of EOC patients, especially those patients that have cancers that are resistant or recalcitrant to platinum agents.
  • the present disclosure provides for combinations and methods that are useful epithelial ovarian cancer (EOC, without limitations, e.g., primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer and endometrial cancer)) therapy.
  • EOC epithelial ovarian cancer
  • the present disclosure in part, relates to a combination of an immunoconjugate and a heterologous chimeric protein comprising a portion of SIRPo and a portion of CD40L adjoined via a linker, both of which enhance the phagocytosis and death of ovarian tumor cells by different and complementary mechanism of actions and promote a robust immune response against EOC.
  • the immunoconjugate binds to FOLR1 (or FRo) present on EOC cells, and induce phagocytosis, CD47 expression, and apoptosis
  • the chimeric protein neutralizes the CD47’s “do not eat me” signal, promoting phagocytosis, and contemporaneously induces an immune response mediated by the CD40 signaling.
  • this combination has anti-tumor effects in a macrophage sparing manner.
  • the present disclosure provides a method for treating an epithelial ovarian cancer (EOC) in a subject in need thereof comprising: administering to the subject a first pharmaceutical composition comprising a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SIRPa(CD172a), wherein the portion is capable of binding a SIRPo(CD172a) ligand, (b) a second domain comprising a portion of the extracellular domain of CD40L, wherein the portion is capable of binding a CD40L receptor, and (c) a linker linking the first domain and the second domain; and administering to the subject a second pharmaceutical composition comprising an immunoconjugate, wherein the immunoconjugate comprises a maytansinoid compound and an anti-FOLR1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementary determining region (CDR)1 sequence of SEQ ID NO: 68, a VH CDR2
  • the first pharmaceutical composition and the second pharmaceutical composition are administered contemporaneously. In embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition is administered. In embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition is administered.
  • the dose of the first pharmaceutical composition is less than the dose of the first pharmaceutical composition administered to a second subject who has not undergone or is not undergoing treatment with the second pharmaceutical composition.
  • the dose of the second pharmaceutical composition administered is less than the dose of the second pharmaceutical composition administered to a second subject who has not undergone or is not undergoing treatment with the first pharmaceutical composition.
  • the subject has an increased chance of survival, without gastrointestinal inflammation and weight loss, and/or a reduction in tumor size or cancer prevalence when compared to a subject who has only undergone or is only undergoing treatment with the first pharmaceutical composition.
  • the subject has an increased chance of survival, without gastrointestinal inflammation and weight loss, and/or a reduction in tumor size or cancer prevalence when compared to a subject who has only undergone or is only undergoing treatment with the second pharmaceutical composition.
  • the present disclosure provides a method for treating a cancer in a subject comprising: administering to the subject a pharmaceutical composition comprising a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SIRPa(CD172a), wherein the portion is capable of binding a SIRPo(CD172a) ligand, (b) a second domain comprising a portion of the extracellular domain of CD40L, wherein the portion is capable of binding a CD40L receptor, and (c) a linker linking the first domain and the second domain; wherein the subject has undergone or is undergoing treatment with a second pharmaceutical composition comprising an immunoconjugate, wherein the immunoconjugate comprises a maytansinoid compound and an anti-FOLR1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementary determining region (CDR)1 sequence of SEQ ID NO: 68, a VH CDR2 sequence of SEQ ID NO: 69,
  • the dose of the pharmaceutical composition administered to the subject is less than the dose of the pharmaceutical composition that is administered to a second subject who has not undergone or is not undergoing treatment with the second pharmaceutical composition.
  • the present disclosure provides a method for treating a cancer in a subject comprising: administering to the subject a second pharmaceutical composition comprising an immunoconjugate, wherein the immunoconjugate comprises a maytansinoid compound and an anti-FOLR1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementary determining region (CDR)1 sequence of SEQ ID NO: 68, a VH CDR2 sequence of SEQ ID NO: 69, and a VH CDR3 sequence of SEQ ID NO: 71 , and a light chain variable region (VL) CDR1 sequence of SEQ ID NO: 65, a VL CDR2 sequence of SEQ ID NO: 66, and a VL CDR3 sequence of SEQ ID NO: 67; wherein the subject has undergone or is undergoing treatment with a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SIRPa(CD172a), wherein the portion is
  • the dose of the pharmaceutical composition provided to the subject is less than the dose of the pharmaceutical composition that is provided to a second subject who has not undergone or is not undergoing treatment with the heterologous chimeric protein.
  • the heterologous chimeric protein comprises a first domain which comprises substantially the entire extracellular domain of SIRPa(CD172a) and/or a second domain which comprises substantially the entire extracellular domain of CD40L.
  • the heterologous chimeric protein comprises: (a) a first domain comprising an extracellular domain of SIRPa(CD172a), (b) a second domain comprising an extracellular domain of CD40L, and (c) a linker comprising a hinge-CH2-CH3 Fc domain.
  • the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence.
  • the linker comprises at least about one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH2-CH3 Fc domain.
  • the linker comprises a hinge-CH2-CH3 Fc domain derived from I gG 1 or I gG4, e.g., human lgG4 or human I gG4.
  • the linker comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  • the first domain comprises an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 57.
  • the second domain comprises an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 58.
  • the heterologous chimeric protein comprises: (a) a first domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 57, (b) a second domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 58, and (c) a linker comprising a hinge-CH2-CH3 Fc domain derived from human lgG4.
  • the heterologous chimeric protein comprises an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 59.
  • the anti-FOLR1 antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 62 and a VL comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 64.
  • the anti-FOLR1 antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 72 and a light chain comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74.
  • the maytansinoid is selected from mertansine (DM1), DM3, and DM4.
  • the maytansinoid is DM4.
  • maytanisonid is linked to the antibody or antigen-binding fragment thereof by a sulfo-SPDB linker.
  • the immunoconjugate comprises 1-10 maytansinoid molecules, 2-8 maytansinoid molecules, 2-5 maytansinoid molecules, or 3-5 maytansinoid molecules, or 3-4 maytansinoid molecules.
  • the immunoconjugate has the following chemical structure:
  • Ab-sulfo-SPDB-DM4 wherein “Ab” represents the anti-FOLR1 antibody or antigen binding fragment thereof.
  • the immunoconjugate comprises 2-5 or 3-4 maytansinoid molecules.
  • the immunoconjugate is administered once every three weeks. In embodiments, the immunoconjugate is administered at a dose in the range of about 1.5 mg/kg to about 4.5 mg/kg adjusted ideal body weight (AIBW), or about 3 mg/kg to about 9 mg/kg AIBW, or about 4.5 mg/kg to about 13.5 mg/kg AIBW, or about 6 mg/kg to about 18 mg/kg AIBW.
  • AIBW adjusted ideal body weight
  • the immunoconjugate is administered at a dose of about 2 mg/kg, or about 3 mg/kg, or about 4 mg/kg, or about 6 mg/kg, or about 8 mg/kg, or about 10 mg/kg, or about 12 mg/kg, or about 14 mg/kg, or about 16 mg/kg, or about 18 mg/kg adjusted ideal body weight (AIBW). In embodiments, the immunoconjugate is administered at a dose of about 3 mg/kg, or about 6 mg/kg adjusted ideal body weight (AIBW).
  • the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 10 mg/kg. In embodiments, the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 0.9 mg/kg, or about 0.5 mg/kg to about 1 .5 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 6 mg/kg, or about 3 mg/kg to about 9 mg/kg, or about 4 mg/kg to about 10 mg/kg. In embodiments, the heterologous chimeric protein is administered at a dose of about 0.3 mg/kg, or about 1 mg/kg, or about 3 mg/kg or about 10 mg/kg.
  • the heterologous chimeric protein is administered at a dose of about 0.3 mg/kg, or about 1 mg/kg, or about 3 mg/kg or about 10 mg/kg), optionally wherein the heterologous chimeric protein is administered once every three weeks (Q3W); and the immunoconjugate is administered at a dose of about 5 mg/kg, or about 6 mg/kg adjusted ideal body weight (AIBW), optionally wherein the immunoconjugate is administered once every three weeks (Q3W).
  • AIBW ideal body weight
  • the heterologous chimeric protein is administered at a dose in the range of about 3 mg/kg to about 10 mg/kg and the immunoconjugate is administered at a dose of about 5 mg/kg or about 6 mg/kg adjusted ideal body weight (AIBW) administered Q3W, wherein the immunoconjugate comprises an average of 3-4 maytansinoid molecules per antibody.
  • AIBW ideal body weight
  • the heterologous chimeric protein is administered once a week or once every two weeks. In embodiments, the heterologous chimeric protein is administered once a week. In embodiments, the heterologous chimeric protein is administered once every two weeks. In embodiments, the heterologous chimeric protein is administered in a biphasic or multiphasic regimen of administration comprising once a week or once every two weeks administrations.
  • the EOC is originated in the ovary, fallopian tube, or peritoneum.
  • the EOC is a primary peritoneal cancer, a primary ovarian cancer, or a primary fallopian tube cancer.
  • the EOC is platinum resistant, relapsed, or refractory.
  • the subject has a histologically confirmed diagnosis of primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer, or high grade serous epithelial ovarian cancer.
  • the subject has received at least about one but no more than two prior systemic lines of anticancer therapy. In embodiments, the subject has received at least about one but no more than three prior systemic lines of anticancer therapy.
  • the subject has received at least about one but no more than five prior systemic lines of anticancer therapy. In embodiments, the subject has received at least about one line of therapy comprising bevacizumab or is medically-ineligible to receive bevacizumab. In embodiments, the subject is relapsed after receiving in a platinum-based therapy. In embodiments, the administration results in a decrease in CA125.
  • the EOC expresses FOLR1 protein.
  • the FOLR1 protein expression has been measured by immunohistochemistry (IHC) in a tumor sample obtained from the subject.
  • IHC immunohistochemistry
  • a staining score of at least about 1 hetero, at least about 1 homo, at least about 2 hetero, at least about 2 homo, or at least about 3 hetero is measured by IHC.
  • at least about 25%, at least about 33%, at least about 50%, at least about 66%, or at least about 75% of cells in the sample obtained from the subject have an IHC score of at least about 2.
  • at least about 25%, at least about 33%, at least about 50%, at least about 66%, or at least about 75% of cells in the sample obtained from the subject have an IHC score of at least about 3.
  • the subject has been determined to be FRa positive as determined by membrane staining visible at less than or equal to 10x microscope objective. In embodiments, the subject has been determined to be FRa positive as determined by immunohistochemistry (IHC). In embodiments, wherein at least about 25% of cells in the sample obtained from the subject have an IHC score of at least about 2. In embodiments, about 25% to about 49% of cells in the sample have an IHC score of at least about 2. In embodiments, about 50% to about 74% of cells in the sample have an IHC score of at least about 2. In embodiments, about 75% to about 100% of cells in the sample have an IHC score of at least about 2.
  • IHC immunohistochemistry
  • the epithelial ovarian cancer exhibits a detectable level surface expression of FOLR1 protein.
  • the level of surface expression of FOLR1 protein is measured by immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS), RNA sequencing, or a combination thereof.
  • the epithelial ovarian cancer (EOC) exhibits a low FOLR1 protein expression as characterized by IHC score (e.g., having at least about 25% of cells to 49% of cells in the sample obtained from the patient having an IHC score of 2).
  • the epithelial ovarian cancer expresses CD47 and/or calreticulin.
  • the EOC expresses CD47, calreticulin and/or CD40.
  • the administration of the immunoconjugate increases the expression and/or activity of CD47, calreticulin, and/or annexin V in an sample of the EOC compared to a control sample comprising an EOC sample from a subject that is obtained prior to the administration of the immunoconjugate or an EOC sample from a different subject that is not been treated with the immunoconjugate, or a healthy tissue from the subject or from a different subject, optionally wherein the expression and/or activity of CD47, calreticulin, and/or annexin V is measured by RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof, optionally wherein the expression and/
  • the immunoconjugate and the heterologous chimeric protein are administered in separate pharmaceutical compositions.
  • the immunoconjugate is administered intravenously or intraperitoneally.
  • the heterologous chimeric protein is administered intravenously.
  • the administration of the heterologous chimeric protein and the immunoconjugate is a first- line therapy. In embodiments, administration is a second-line therapy. In embodiments, the administration of the heterologous chimeric protein and the immunoconjugate is a third-line or later than third line therapy.
  • the subject has previously been treated with a platinum compound, a taxane, bevacizumab, a PARP inhibitor, or a combination thereof.
  • the EOC is primary platinum refractory.
  • the EOC is platinum resistant.
  • the EOC is metastatic or advanced.
  • the administration of the heterologous chimeric protein and the immunoconjugate produces a greater therapeutic benefit than administration of the heterologous chimeric protein alone or the immunoconjugate alone. In embodiments, the administration of the heterologous chimeric protein and the immunoconjugate does not produce more toxicity than the toxicity produced by the administration of the heterologous chimeric protein alone or the immunoconjugate alone.
  • the administration of the heterologous chimeric protein and the immunoconjugate increases or stimulated the level of phagocytosis of EOC cells compared to the level of phagocytosis of EOC cells prior to the administration of the immunoconjugate or an EOC sample from a different subject that is not been treated with the heterologous chimeric protein and the immunoconjugate.
  • the administration of the heterologous chimeric protein and the immunoconjugate does not stimulate apoptosis of macrophages compared to or drugs that macrophage apoptosis (e.g., bisphosphonates).
  • the present disclosure provides a method of treating a patient having epithelial ovarian cancer (EOC) cancer comprising administering to said patient in need thereof a first pharmaceutical composition comprising about 0.3 mg/Kg to about 10 mg/Kg of a heterologous chimeric protein wherein the heterologous chimeric protein comprises a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain is capable of binding a SIRPo(CD172a) ligand and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 57, (b) is a second domain is capable of binding a CD40L receptor and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 58, and (c) is a linker adjoining (a) and (b) and comprising a hinge-CH2-CH3 Fc domain derived from human lg
  • the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 0.9 mg/kg, or about 0.5 mg/kg to about 1 .5 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 6 mg/kg, or about 3 mg/kg to about 9 mg/kg, or about 4 mg/kg to about 10 mg/kg.
  • the immunoconjugate is administered at a dose in the range of about 1 .5 mg/kg to about 4.5 mg/kg adjusted ideal body weight (AIBW), or about 3 mg/kg to about 9 mg/kg AIBW, or about 4.5 mg/kg to about 13.5 mg/kg AIBW, or about 6 mg/kg to about 18 mg/kg AIBW.
  • AIBW adjusted ideal body weight
  • the first pharmaceutical composition and the second pharmaceutical composition are administered contemporaneously. In embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition is administered. In embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition is administered.
  • the linker comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  • the heterologous chimeric protein comprises an amino acid sequence that is at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 59.
  • the immunoconjugate comprises 1-10, 2-8, 2-5, or 3-4 maytansinoids.
  • the immunoconjugate comprises 2-5 or 3-4 maytansinoids.
  • the immunoconjugate has the following chemical structure: wherein “Ab” represents the anti-FOLR 1 antibody or antigen binding fragment thereof.
  • the EOC has originated in the ovary cancer, fallopian tube, or peritoneum cancer.
  • the EOC is primary peritoneal cancer.
  • the EOC is primary fallopian tube cancer.
  • the EOC is epithelial ovarian cancer.
  • the EOC is platinum resistant, relapsed, or refractory.
  • the patient has a histologically confirmed diagnosis of primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer, or high grade serous epithelial ovarian cancer.
  • the patient has received at least about one but no more than three prior systemic lines of anticancer therapy.
  • the patient has received at least about one line of therapy comprising bevacizumab or is medically-ineligible to receive bevacizumab.
  • FIG. 1 shows a schematic illustration of a hexameric form of the SIRPo-Fc-CD40L chimeric protein (SL- 172154), which is includes extracellular domains each of SIRPo and CD40L, joined by a linker that comprises an Fc domain.
  • FIG. 2A and FIG. 2B show the flow phenotyping of KB ovarian cancer cells when grown in the presence of indicated concentrations of Mirvetuximab for 1, 3, 6 or 24 hours.
  • FIG. 2A shows the surface expression of calreticulin, CD40 and Mirvetuximab-bound folate receptor a (FRa).
  • FIG. 2B shows the surface expression of annexin V (a marker of apoptosis) and CD47.
  • FIG. 3A and FIG. 3B show the flow phenotyping of IGROV-1 human ovarian carcinoma cells when grown in the presence of indicated concentrations of Mirvetuximab for 1 , 3, 6 or 24 hours.
  • FIG. 3A shows the surface expression of calreticulin, CD40 and Mirvetuximab-bound folate receptor a (FRa).
  • FIG. 3B shows the surface expression of annexin V (a marker of apoptosis) and CD47.
  • FIG. 4A and FIG. 4B show the flow phenotyping of OV90 human ovarian carcinoma cells when grown in the presence of indicated concentrations of Mirvetuximab for 1 , 3, 6 or 24 hours.
  • FIG. 4A shows the surface expression of calreticulin, CD40 and Mirvetuximab-bound folate receptor a (FRa).
  • FIG. 4B shows the surface expression of annexin V (a marker of apoptosis) and CD47.
  • FIG. 5A and FIG. 5B show the flow phenotyping of SKOV-3 human ovarian carcinoma cells when grown in the presence of indicated concentrations of Mirvetuximab for 1 , 3, 6 or 24 hours.
  • FIG. 5A shows the surface expression of calreticulin, CD40 and Mirvetuximab-bound folate receptor a (FRa).
  • FIG. 5B shows the surface expression of annexin V (a marker of apoptosis) and CD47.
  • FIG. 6A to FIG. 6M show the flow phenotyping of various ovarian cancer cell lines as determined by flow cytometry.
  • FIG. 6G shows the FRa receptor occupancy as determined by flow cytometry after the treatment with 3 hours in culture in the presence of the indicated concentration of Mirvetuximab (ADC) in the ovarian cancer cell lines KB, IGROV-1, OV90, SKOV-3 and MES-0V3.
  • the cell surface expression of CD47 (FIG.
  • FIG. 7A to FIG. 7C show the baseline expression analysis CD47 (FIG. 7A) and calreticulin (FIG. 7B) and the Mirvetuximab-bound folate receptor a (FRa, FIG. 7C) in a battery of ovarian cancer cell lines.
  • FIG. 8 shows a summary of flow phenotyping of the ovarian cancer cell lines KB, IGROV-1, OV90 and SKOV- 3. CD47 and CD40.
  • the surface expression were assessed by flow cytometry on at baseline, calreticulin (CALR), Annxin V, and FRa were assessed before and after incubation of tumor cells with Mirvetuximab.
  • CAR calreticulin
  • Annxin V Annxin V
  • FRa were assessed before and after incubation of tumor cells with Mirvetuximab.
  • Mirvetuximab was used as the cell surface detection reagent.
  • FIG. 9A and FIG. 9B show the results of a phagocytosis assay using macrophages co-cultured with KB (FIG. 9A) or OV90 (FIG. 9B) ovarian cancer cells, in the presence of 1 pg/ml or 10 pg/ml Mirvetuximab, as compared to vehicle only control.
  • the extent of apoptosis in the presence of the vehicle only control is indicated by a dotted line.
  • FIG. 10Ato FIG. 10D show the results of a phagocytosis assay using macrophages co-cultured with KB (FIG. 10A), IGROV-1 (FIG. 10B), OV90 (FIG. 10C), or MES-OV3 (FIG. 10D) ovarian cancer cells, in the presence of 50 pg/ml of the SIRPa-Fc-CD40L chimeric protein (SL-172154), 10 pg/ml Mirvetuximab, or a combination of the two, as compared to vehicle only control. The extent of apoptosis in the presence of the vehicle only control is indicated by a dotted line.
  • FIG. 11 A and FIG. 11 B show the macrophage response.
  • FIG. 11 A shows the annexin V expression in human in human PBMC-derived M1 macrophages when cultured in the presence of 1 pg/ml Mirvetuximab, 10 pg/ml of the SIRPa-Fc-CD40L chimeric protein (SL-172154), or a combination of the two.
  • FIG. 11 B shows the annexin V expression in human in human PBMC-derived M1 macrophages when cultured in the presence of 10 pg/ml Mirvetuximab, 50 pg/ml of the SIRPa-Fc-CD40L chimeric protein (SL-172154), or a combination of the two
  • FIG. 12 shows a schematic representation of the initial clinical development strategy of combination of the SIRPa-Fc-CD40L chimeric protein (SL-172154) and mirvetuximab in ovarian cancer.
  • dose levels ranging from about 1 mg/Kg to about 10 mg/Kg, of the SIRPa-Fc-CD40L chimeric protein (SL-172154).
  • the present disclosure is based, in part, on the discovery that the combination of a heterologous chimeric protein comprising a portion of SIRPa and a portion of CD40L adjoined via a linker and an immunoconjugate enhanced the phagocytosis of ovarian tumor cell lines, and that the majority of ovarian cancer cell lines that were tested show high level of expression of folate receptor alpha (FRa) and CD47, which are receptors for Mirvetuximab and the SIRPa part of SL-172154. Moreover, as shown herein, this combination has anti-tumor effects in a macrophage sparing manner. Accordingly, in one aspect, the present disclosure provides combinations of an anti-FOLR1 immunoconjugate with chimeric protein comprising SIRPa and CD40L and the use of the combinations in the treatment of EOC.
  • FRa folate receptor alpha
  • CD47 which are receptors for Mirvetuximab and the SIRPa part of SL-172154.
  • this combination has anti-
  • the present disclosure in part, relates to a combination of an immunoconjugate and a heterologous chimeric protein comprising a portion of SIRPa and a portion of CD40L adjoined via a linker, both of which enhance the phagocytosis and death of ovarian tumor cells by different and complementary mechanism of actions and promote a robust immune response against EOC.
  • the immunoconjugate present on EOC cells (without limitations, e.g., primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer and endometrial cancer)), and induces phagocytosis, CD47 and apoptosis
  • the chimeric protein neutralizes the CD47’s “do not eat me” signal, promoting phagocytosis, and simultaneously induces an immune response mediated by the CD40 signaling.
  • FOLR1 refers to any native human FOLR1 polypeptide, unless otherwise indicated.
  • FOLR1 is also referred to as “human folate receptor 1 ,” “folate receptor alpha (FR-a),” and “FRa”.
  • the term “FOLR1” encompasses “full-length,” unprocessed FOLR1 polypeptide as well as any form of FOLR1 polypeptide that results from processing within the cell.
  • the term also encompasses naturally occurring variants of FOLR1, e.g., those encoded by splice variants and allelic variants.
  • the FOLR1 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
  • FOLR1 can be used to refer to a nucleic acid that encodes a FOLR1 polypeptide.
  • Human FOLR1 sequences are known and include, for example, the sequences publicly available at UniProtKB Accession No. P15328 (including isoforms).
  • human FOLR1 refers to FOLR1 comprising the sequence of SEQ ID NO: 60.
  • antibody means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least about one antigen recognition site within the variable region of the immunoglobulin molecule.
  • a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least about one antigen recognition site within the variable region of the immunoglobulin molecule.
  • the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity.
  • An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.
  • antibody fragment refers to a portion of an intact antibody.
  • An “antigen-binding fragment” refers to a portion of an intact antibody that binds to an antigen.
  • An antigen-binding fragment can contain the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies.
  • anti-FOLR1 antibody or “an antibody that binds to FOLR1” refers to an antibody that is capable of binding FOLR1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting FOLR1 (e.g., the huMov19 (M9346A) antibody).
  • the extent of binding of an anti-FOLR1 antibody to an unrelated, non-FOLR1 protein can be less than about 10% of the binding of the antibody to FOLR1 as measured, e.g., by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • line of treatment or “line of therapy” refer to a therapeutic regimen that can include but is not limited to surgery, radiation therapy, chemotherapy, differentiating therapy, biotherapy, immune therapy, or the administration of one or more anti-cancer agents (e.g., a cytotoxic agent, an anti-proliferative compound, and/or an angiogenesis inhibitor).
  • anti-cancer agents e.g., a cytotoxic agent, an anti-proliferative compound, and/or an angiogenesis inhibitor.
  • first-line treatment refers to an initial treatment for a particular condition, e.g., a given type and stage of cancer, and can e.g., be the preferred or standard treatment. These treatments differ from “second-line” therapies, which are tried when a first-line therapy does not work adequately. ‘Third-line” therapies are tried when a first-line therapy and a second-line therapy do not work adequately.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a first-line therapy e.g., a second-line therapy
  • a third-line therapy e.g., in patients with platinum sensitive or platinum resistant primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer and endometrial cancer.
  • a FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) provided herein can be given as a line of therapy in patients having received no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, or no more than 6 lines of therapy.
  • a FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) provided herein can be given as an adjuvant therapy a neoadjuvant therapy, or a maintenance therapy.
  • adjuvant therapy refers to systemic therapy given after surgery.
  • adjuvant therapy in the broadest sense, is treatment given in addition to the primary therapy to kill any cancer cells that may have spread, even if the spread cannot be detected by radiologic or laboratory tests.
  • neoadjuvant therapy refers to systemic therapy given prior to surgery.
  • maintenance therapy refers to therapy that is given to help keep cancer from coming back after it has disappeared following the initial therapy.
  • IMGN853 also known as mirvetuximab soravtansine or mirvetuximab refers to the immunoconjugate described herein containing the huMov19 (M9346A) antibody, the sulfoSPDB linker, and the DM4 maytansinoid.
  • the huMovI 9 (M9346A) antibody is an anti-FO L R 1 antibody comprising the variable heavy chain sequence SEQ ID NO: 62 and the variable light chain sequence SEQ ID NO: 64.
  • DM4 refers to N2’-deacetyl- N2’-(4-mercapto-4-methyl-1 -oxopentyl) maytansine.
  • SulfoSPDB refers to the N-succinimidyl 4-(2- pyridyldithio)-2-sulfobutanoate) linker.
  • the huMov19 (M9346A) antibody comprises an average of about 3 to about 5 DM4 per antibody.
  • a “monoclonal” antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigenbinding fragment population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants.
  • the term “monoclonal” antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site.
  • “monoclonal” antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.
  • humanized antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences.
  • humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (“CDR grafted”) (Jones etal., Nature 321 :522-525 (1986); Riechmann etal., Nature 332:323-327 (1988); Verhoeyen ef al., Science 239:1534-1536 (1988)), each of which is incorporated by reference in its entirety.
  • CDR complementary determining region
  • the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody or fragment from a non-human species that has the desired specificity, affinity, and capability.
  • the humanized antibody or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non- human residues to refine and optimize antibody or antigen-binding fragment thereof specificity, affinity, and/or capability.
  • the humanized antibody or antigen-binding fragment thereof will comprise substantially all of at least about one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody or antigenbinding fragment thereof can also comprise at least about a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539; Roguska et al., Proc. Natl. Acad.
  • a “humanized antibody” is a resurfaced antibody.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions.
  • FR framework regions
  • CDRs complementarity determining regions
  • the CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.
  • CDRs There are at least about two techniques for determining CDRs: (1) an approach based on crossspecies sequence variability (/.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991 , National Institutes of Health, Bethesda Md.), “Kabat”); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al, J. Molec. Biol. 273: 927-948 (1997)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.
  • Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. (5th Ed., 1991 , National Institutes of Health, Bethesda, Md.) (“Kabat”).
  • amino acid position numbering refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al. (Sequences of Immunological Interest. (5th Ed., 1991 , National Institutes of Health, Bethesda, Md.), “Kabat”). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.
  • the Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)).
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • chimeric antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species.
  • the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigenbinding fragments thereof derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.
  • epitopes or “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody.
  • the antigen is a polypeptide
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing.
  • An epitope typically includes at least about 3, and more usually, at least about 5 or 8-10 amino acids in a unique spatial conformation.
  • binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • binding affinity refers to a stronger binding between a molecule and its binding partner.
  • Or better when used herein refers to a stronger binding, represented by a smaller numerical Kd value.
  • an antibody which has an affinity for an antigen of “0.6 nM or better” the antibody's affinity for the antigen is ⁇ 0.6 nM, /.e., 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.
  • an antibody binds to an epitope via its antigen binding domain and that the binding entails some complementarity between the antigen binding domain and the epitope, or a heterologous chimeric protein binds a ligand/receptor and that the binding entails some complementarity between the heterologous chimeric protein and the ligand/receptor.
  • an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope.
  • telomere binding domain present in the heterologous chimeric protein more readily than it would bind to a random, unrelated ligand/receptor.
  • preferentially binds it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope.
  • an antibody which “preferentially binds” to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.
  • An antibody is said to “competitively inhibit” binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope.
  • Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays.
  • An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least about 90%, at least about 80%, at least about 70%, at least about 60%, or at least about 50%.
  • substantially similar denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values).
  • the difference between said two values can be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference/comparator antibody.
  • a polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • substantially pure refers to material which is at least about 50% pure (/.e., free from contaminants), at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure.
  • Immunoconjugates can also be defined by the generic formula in reverse order: A-L-C.
  • a “linker” is any chemical moiety that is capable of linking a compound, usually a drug (such as a maytansi noid), to a cell-binding agent (such as an anti FOLR1 antibody or a fragment thereof) in a stable, covalent manner.
  • Linkers can be susceptible to or be substantially resistant to, e.g., disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups and thioether groups.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • examples of cancer include primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer and endometrial cancer.
  • Another example of cancer is endometrial cancer.
  • the cancer can be a cancer that expresses FOLR1 (“FOLR1 -expressing cancer” or “FRa positive” cancer).
  • cancer cell refers to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).
  • tumorigenic stem cells cancer stem cells.
  • tumorigenic stem cells cancer stem cells.
  • an “advanced” cancer is one which has spread outside the site or organ of origin, either by local invasion or metastasis.
  • the term “advanced” cancer includes both locally advanced and metastatic disease.
  • Metalstatic cancer refers to cancer that has spread from one part of the body to another part of the body.
  • a “refractory” cancer is one that progresses even though an anti-tumor treatment, such as a chemotherapy, is administered to the cancer patient.
  • An example of a refractory cancer is one which is platinum refractory.
  • a patient is “platinum-refractory” if the patient does not respond to platinum-based therapy and shows progression during the course of therapy or within 4 weeks after the last dose.
  • Platinum-resistant patients progress within 6 months of platinum-based therapy. “Partially platinumsensitive” patients progress between 6 and 12 months of platinum-based therapy. “Platinum-sensitive” patients progress within an interval of 12 months or more.
  • a “recurrent” cancer is one that has regrown, either at the initial site or at a distant site, after a response to initial therapy.
  • subject refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • subject and “patient” are used interchangeably herein in reference to a human subject.
  • a “relapsed” patient is one who has signs or symptoms of cancer after remission.
  • the patient has relapsed after adjuvant or neoadjuvant therapy.
  • Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) or consecutive administration in any order.
  • the combination therapy can provide “synergy” and prove “synergistic”, /.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.
  • a synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered serially, by alternation, or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes.
  • a synergistic combination produces effects that are greater than the additive effects of the individual components of the combination.
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • the formulation can be sterile.
  • an “effective amount” of an antibody, immunoconjugate, a heterologous chimeric protein, or other drug as disclosed herein is an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • therapeutically effective amount refers to an amount of an antibody, immunoconjugate, or other drug effective to “treat” a disease or disorder in a subject or mammal.
  • the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size or burden; inhibit (/.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (/.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some extent, tumor growth; relieve to some extent one or more of the symptoms associated with the cancer; and/or result in a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), a decrease in CA125 in the case of EOC or any combination thereof. See the definition herein of “treating”.
  • PFS progression-free survival
  • the term “respond favorably” generally refers to causing a beneficial state in a subject.
  • the term refers to providing a therapeutic effect on the subject.
  • Positive therapeutic effects in cancer can be measured in a number of ways (See, W.A. Weber, J. Nucl. Med. 50:1 S-10S (2009)).
  • tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess therapeutic efficacy of an anti-cancer therapeutic.
  • Log 10 Cell Kill (LCK) can be used to quantify the tumor cell kill.
  • the ability to reduce tumor volume may be assessed, for example, by measuring a %T/C value, which is the median tumor volume of treated subjects divided by the median tumor volume of the control subjects.
  • T/C ⁇ 42% is the minimum level of anti-tumor activity.
  • a favorable response can be assessed, for example, by increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), a decrease in CA125 in the case of EOC, or any combination thereof.
  • a “decrease in CA125 levels” can be assessed according to the Gynecologic Cancer Intergroup (GCIG) guidelines. For example, CA125 levels can be measured prior to treatment to establish a baseline CA125 level. CA125 levels can be measured one or more times during or after treatment, and a reduction in the CA125 levels over time as compared to the baseline level is considered a decrease in CA125 levels.
  • GCIG Gynecologic Cancer Intergroup
  • AIBW adjusted ideal body weight
  • AjBW or ADJ adjusted body weight
  • FOLR1 increased expression or “overexpression” of FOLR1 in a particular tumor, tissue, or cell sample refers to FOLR1 (a FOLR1 polypeptide or a nucleic acid encoding such a polypeptide) that is present at a level higher than that which is present in a healthy or non-diseased (native, wild type) tissue or cells of the same type or origin.
  • Such increased expression or overexpression can be caused, for example, by mutation, gene amplification, increased transcription, increased translation, or increased protein stability.
  • a subject is successfully “treated” for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor burden; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non- tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), a decrease in CA125 in the case of EOC,
  • PFS
  • polypeptide “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the polypeptides of this invention are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.
  • a “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the FOLR1 or VEGF to which the polypeptide or antibody binds.
  • Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).
  • the methods of the present disclosure comprise methods for treating cancer, which, in embodiments, comprise administering a pharmaceutical composition comprising a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SIRPa(CD172a), (b) a second domain comprising a portion of the extracellular domain of CD40L, and (c) a linker linking the first domain and the second domain.
  • a pharmaceutical composition comprising a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SIRPa(CD172a), (b) a second domain comprising a portion of the extracellular domain of CD40L, and (c) a linker linking the first domain and the second domain.
  • the present disclosure relates to a method for treating a cancer in a subject in need thereof comprising: a) a first domain comprising a portion of the extracellular domain of SI RPa(CD172a), wherein the portion is capable of binding a SI RPa(CD172a) ligand, (b) a second domain comprising a portion of the extracellular domain of CD40L, wherein the portion is capable of binding a CD40L receptor, and (c) a linker linking the first domain and the second domain.
  • Transmembrane proteins typically consist of an extracellular domain, one or a series of transmembrane domains, and an intracellular domain.
  • the extracellular domain of a transmembrane protein is responsible for interacting with a soluble receptor or ligand or membrane-bound receptor or ligand (/.e., a membrane of an adjacent cell) in the extracellular environment.
  • the trans-membrane domain(s) is responsible for localizing the transmembrane protein to the plasma membrane.
  • the intracellular domain of a transmembrane protein is responsible for coordinating interactions with cellular signaling molecules to coordinate intracellular responses with the extracellular environment (or visa-versa).
  • the chimeric proteins useful in the methods disclosed herein eliminate or reduce side effects associated with disrupting the SIRP1 a/CD47 signaling axis.
  • the present chimeric proteins or methods utilizing the same eliminate or reduce hematological adverse effects.
  • the present chimeric proteins or methods utilizing the same eliminate or reduce the extent of reductions in the number of circulating red blood cells and platelets, hemolysis, hemagglutination, thrombocytopenia, and/or anemia.
  • the present chimeric proteins or methods utilizing the same demonstrate comparatively less hematological adverse effects than an anti-CD47 antibody.
  • an extracellular domain refers to a portion of a transmembrane protein which is sufficient for binding to a ligand or receptor and is effective in transmitting a signal to a cell.
  • an extracellular domain is the entire amino acid sequence of a transmembrane protein which is normally present at the exterior of a cell or of the cell membrane.
  • an extracellular domain is that portion of an amino acid sequence of a transmembrane protein which is external of a cell or of the cell membrane and is needed for signal transduction and/or ligand binding as may be assayed using methods know in the art (e.g., in vitro ligand binding and/or cellular activation assays).
  • an extracellular domain refers to a portion of a transmembrane protein which is sufficient for binding to a ligand or receptor and is effective in transmitting a signal to a cell.
  • an extracellular domain is the entire amino acid sequence of a transmembrane protein which is normally present at the exterior of a cell or of the cell membrane.
  • an extracellular domain is that portion of an amino acid sequence of a transmembrane protein which is external of a cell or of the cell membrane and is needed for signal transduction and/or ligand binding as may be assayed using methods know in the art (e.g., in vitro ligand binding and/or cellular activation assays).
  • Type I transmembrane proteins which have an extracellular amino terminus and an intracellular carboxy terminus
  • Type II transmembrane proteins which have an extracellular carboxy terminus and an intracellular amino terminus.
  • Type I and Type II transmembrane proteins can be either receptors or ligands.
  • SIRPa(CD172a) which is a Type I transmembrane protein
  • the amino terminus of the protein faces outside the cell, and therefore contains the functional domains that are responsible for interacting with other binding partners (without limitation, e.g., CD47) in the extracellular environment.
  • CD40L which is a Type II transmembrane protein
  • the carboxy terminus of the protein faces outside the cell, and therefore contains the functional domains that are responsible for interacting with other binding partners (without limitation, e.g., CD40) in the extracellular environment.
  • these two types of transmembrane proteins have opposite orientations to each other relative to the cell membrane.
  • Chimeric proteins used in methods of the present disclosure comprise an extracellular domain of SIRPa(CD172a), and an extracellular domain of CD40L.
  • a heterologous chimeric protein used in a method of the present disclosure comprises, at least about, a first domain comprising the extracellular domain of SIRPa(CD172a), which is connected - directly or via a linker - to a second domain comprising the extracellular domain of CD40L.
  • the domains are linked in an amino-terminal to carboxy-terminal orientation, the first domain is located on the “left”’ side of the chimeric protein and is “outward facing” and the second domain is located on “right” side of the chimeric protein and is “outward facing”.
  • first and second domains are envisioned, e.g., the first domain is inward facing and the second domain is outward facing, the first domain is outward facing and the second domain is inward facing, and the first and second domains are both inward facing.
  • both domains are “inward facing”
  • the chimeric protein would have an amino-terminal to carboxy-terminal configuration comprising an extracellular domain of a Type II transmembrane protein, a linker, and an extracellular domain of Type I transmembrane protein.
  • the heterologous chimeric protein comprises: (a) a first domain comprising a portion of the extracellular domain of SIRPa(CD172a), wherein the portion is capable of binding a SIRPa(CD172a) ligand, (b) a second domain comprising a portion of the extracellular domain of CD40L, wherein the portion is capable of binding a CD40L ligand, and (c) a linker linking the first domain and the second domain.
  • a heterologous chimeric protein comprises a first domain which comprises substantially the entire extracellular domain of SIRPa(CD172a), and/or the second domain which comprises substantially the entire extracellular domain of CD40L.
  • the first domain which comprises substantially the entire extracellular domain of SIRPa(CD172a).
  • the second domain which comprises substantially the entire extracellular domain of CD40L.
  • the first domain comprises a portion of Signal regulatory protein a (SIRPa). In embodiments, the first domain comprises the extracellular domain of SIRPa. In embodiments, the first domain comprises the CD47-binding portion of SIRPa.
  • SIRPa Signal regulatory protein a
  • a heterologous chimeric protein used in methods of the present disclosure comprises the extracellular domain of human SIRPa(CD172a) which comprises the following amino acid sequence:
  • a heterologous chimeric protein used in methods of the present disclosure comprises a variant of the extracellular domain of SIRPa(CD172a).
  • the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%
  • the variant of the extracellular domain of SIRPa(CD172a) has at least about 95% sequence identity with SEQ ID NO: 57
  • a heterologous chimeric protein used in methods of the present disclosure comprises the extracellular domain of human CD40L which comprises the following amino acid sequence:
  • a heterologous chimeric protein used in methods of the present disclosure comprises a variant of the extracellular domain of CD40L.
  • the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least
  • the variant of the extracellular domain of CD40L has at least about 95% sequence identity with SEQ ID NO: 58
  • the chimeric protein may comprise an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences disclosed herein.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. “Conservative substitutions” may be made, for instance, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
  • the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • conservative substitutions are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
  • the substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, /V-formylmethionine 0-alanine, GABA and 6-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, £-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclo
  • Mutations may also be made to the nucleotide sequences of the chimeric proteins by reference to the genetic code, including taking into account codon degeneracy.
  • a heterologous chimeric protein is capable of binding murine ligand(s)/receptor(s).
  • a heterologous chimeric protein is capable of binding human ligand(s)/receptor(s).
  • each extracellular domain (or variant thereof) of the chimeric protein binds to its cognate receptor or ligand with a KD of about 1 nM to about 5 nM, for example, about 1 nM, about 1 .5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM.
  • the chimeric protein binds to a cognate receptor or ligand with a KD of about 5 nM to about 15 nM, for example, about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM, about 9.5 nM, about 10 nM, about 10.5 nM, about 11 nM, about 11.5 nM, about 12 nM, about 12.5 nM, about 13 nM, about 13.5 nM, about 14 nM, about 14.5 nM, or about 15 nM.
  • each extracellular domain (or variant thereof) of the chimeric protein binds to its cognate receptor or ligand with a KD of less than about 1 pM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 150 nM, about 130 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, or about 5 nM, or about 1 nM (as measured, for example, by surface plasmon resonance or biolayer interferometry).
  • the chimeric protein binds to human CD47 and/or CD40 with a KD of less than about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (as measured, for example, by surface plasmon resonance or biolayer interferometry).
  • a variant of an extracellular domain is capable of binding the receptor/ligand of a native extracellular domain.
  • a variant may include one or more mutations in an extracellular domain which do not affect its binding affinity to its receptor/ligand; alternately, the one or more mutations in an extracellular domain may improve binding affinity for the receptor/ligand; or the one or more mutations in an extracellular domain may reduce binding affinity for the receptor/ligand, yet not eliminate binding altogether.
  • the one or more mutations are located outside the binding pocket where the extracellular domain interacts with its receptor/ligand. In embodiments, the one or more mutations are located inside the binding pocket where the extracellular domain interacts with its receptor/ligand, as long as the mutations do not eliminate binding altogether.
  • the chimeric protein exhibits enhanced stability, high-avidity binding characteristics, prolonged off-rate for target binding and protein half-life relative to single-domain fusion protein or antibody controls.
  • Chimeric proteins of the present disclosure and/or chimeric proteins used in methods of the present disclosure have a first domain which is sterically capable of binding its ligand/receptor and/or a second domain which is sterically capable of binding its ligand/receptor. This means that there is sufficient overall flexibility in the chimeric protein and/or physical distance between an extracellular domain (or a portion thereof) and the rest of the chimeric protein such that the ligand/receptor binding domain of the extracellular domain is not sterically hindered from binding its ligand/receptor.
  • This flexibility and/or physical distance may be normally present in the extracellular domain(s), normally present in the linker, and/or normally present in the chimeric protein (as a whole).
  • the chimeric protein may be modified by including one or more additional amino acid sequences (e.g., the joining linkers described below) or synthetic linkers (e.g., a polyethylene glycol (PEG) linker) which provide additional slack needed to avoid steric hindrance.
  • additional amino acid sequences e.g., the joining linkers described below
  • synthetic linkers e.g., a polyethylene glycol (PEG) linker
  • the chimeric protein used in a method of the present disclosure comprises a linker.
  • the linker comprising at least about one cysteine residue capable of forming a disulfide bond.
  • the at least about one cysteine residue is capable of forming a disulfide bond between a pair (or more) of chimeric proteins.
  • disulfide bond forming is responsible for maintaining a useful multimeric state of chimeric proteins. This allows for efficient production of the chimeric proteins; it allows for desired activity in vitro and in vivo.
  • stabilization in a linker region including one or more disulfide bonds provides for improved chimeric proteins that can maintain a stable and producible multimeric state.
  • the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence.
  • the linker is derived from naturally-occurring multi-domain proteins or is an empirical linker as described, for example, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contents of which are hereby incorporated by reference.
  • the linker may be designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et. al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.
  • the linker comprises a polypeptide.
  • the polypeptide is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long.
  • the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.
  • the linker is flexible.
  • the linker is rigid.
  • the linker is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines).
  • the linker comprises a hinge region of an antibody (e.g., of IgG, IgA, IgD, and I g E, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1 , and lgA2)).
  • the hinge region found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space.
  • the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses.
  • the hinge region of lgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges.
  • lgG2 has a shorter hinge than lgG1 , with 12 amino acid residues and four disulfide bridges.
  • the hinge region of lgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the I gG2 molecule.
  • I gG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the lgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix.
  • the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility.
  • the elongated hinge in I gG3 is also responsible for its higher molecular weight compared to the other subclasses.
  • the hinge region of lgG4 is shorter than that of lgG1 and its flexibility is intermediate between that of I gG1 and lgG2.
  • the linker may be derived from human lgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.
  • the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region.
  • the upper hinge region includes amino acids from the carboxyl end of CHI to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains.
  • the length of the upper hinge region correlates with the segmental flexibility of the antibody.
  • the core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id.
  • the core hinge region of wild-type human lgG1 contains the sequence CPPC (SEQ ID NO: 24) which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility.
  • the present linker comprises, one, or two, or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1 and lgA2)).
  • the hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment.
  • lgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin.
  • the linker of the present disclosure comprises one or more glycosylation sites.
  • the linker comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1 and lgA2)).
  • an antibody e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1 and lgA2)).
  • the linker comprises a hinge- CH2-CH3 Fc domain derived from lgG4. In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human lgG4. In embodiments, the linker comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3, e.g., at least about 95% identical to the amino acid sequence of SEQ ID NO: 2. In embodiments, the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NO: 4 to SEQ ID NO: 50 (or a variant thereof).
  • the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NO: 4 to SEQ ID NO: 50 (or a variant thereof); wherein one joining linker is N terminal to the hinge-CH2-CH3 Fc domain and another joining linker is C terminal to the hinge-CH2-CH3 Fc domain.
  • the linker comprises a hinge-CH2-CH3 Fc domain derived from a human lgG1 antibody.
  • the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn).
  • the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the chimeric proteins used in methods of the present disclosure.
  • the Fc domain in a linker contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311 , 416, 428, 433 or 434 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference), or equivalents thereof.
  • the amino acid substitution at amino acid residue 250 is a substitution with glutamine.
  • the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine.
  • the amino acid substitution at amino acid residue 254 is a substitution with threonine.
  • the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine.
  • the amino acid substitution at amino acid residue 308 is a substitution with threonine.
  • the amino acid substitution at amino acid residue 309 is a substitution with proline.
  • the amino acid substitution at amino acid residue 311 is a substitution with serine.
  • the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine.
  • the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine.
  • the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine.
  • the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine.
  • the amino acid substitution at amino acid residue 416 is a substitution with serine.
  • the amino acid substitution at amino acid residue 428 is a substitution with leucine.
  • the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine.
  • the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine.
  • the Fc domain linker (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference).
  • the IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation.
  • the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation.
  • the IgG constant region includes an YTE and KFH mutation in combination.
  • the linker comprises an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference).
  • Illustrative mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A.
  • the IgG constant region comprises a M428L/N434S mutation or LS mutation. In embodiments, the IgG constant region comprises a T250Q/M428L mutation or QL mutation. In embodiments, the IgG constant region comprises an N434A mutation. In embodiments, the IgG constant region comprises a T307A/E380A/N434A mutation or AAA mutation. In embodiments, the IgG constant region comprises an I253A/H310A/H435A mutation or IHH mutation. In embodiments, the IgG constant region comprises a H433K/N434F mutation. In embodiments, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination.
  • An illustrative Fc stabilizing mutant is S228P.
  • Illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311 S and the present linkers may comprise 1 , or 2, or 3, or 4, or 5 of these mutants.
  • the chimeric protein binds to FcRn with high affinity.
  • the chimeric protein may bind to FcRn with a KD of about 1 nM to about 80 nM.
  • the chimeric protein may bind to FcRn with a KD of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 n
  • the chimeric protein may bind to FcRn with a KD of about 9 nM. In embodiments, the chimeric protein does not substantially bind to other Fc receptors (/.e., other than FcRn) with effector function.
  • the Fc domain in a linker has the amino acid sequence of SEQ ID NO: 1 (see Table 1, below), or at least about 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto.
  • mutations are made to SEQ ID NO: 1 to increase stability and/or half-life.
  • the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 2 (see Table 1 , below), or at least about 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto.
  • the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 3 (see Table 1, below), or at least about 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto.
  • one or more joining linkers may be employed to connect an Fc domain in a linker (e.g., one of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or at least about 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto) and the extracellular domains.
  • a linker e.g., one of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or at least about 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto
  • any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or variants thereof may connect an extracellular domain as disclosed herein and an Fc domain in a linker as disclosed herein.
  • any one of SEQ ID NO: 4 to SEQ ID NO: 50, or variants thereof are located between an extracellular domain as disclosed herein and an Fc
  • a linker may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%,
  • first and second joining linkers may be different or they may be the same.
  • linker comprising at least about a part of an Fc domain in a heterologous chimeric protein, helps avoid formation of insoluble and, likely, non-functional protein concatenated oligomers and/or aggregates. This is in part due to the presence of cysteines in the Fc domain which are capable of forming disulfide bonds between chimeric proteins.
  • a heterologous chimeric protein may comprise one or more joining linkers, as disclosed herein, and lack an Fc domain linker, as disclosed herein.
  • first and/or second joining linkers are independently selected from the amino acid sequences of SEQ ID NO: 4 to SEQ ID NO: 50 and are provided in Table 1 below:
  • the joining linker substantially comprises glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines).
  • the joining linker is (Gly 4 Ser) n , where n is from about 1 to about 8, e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO: 25 to SEQ ID NO: 32, respectively).
  • the joining linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 33).
  • X designating any amino acid, e.g., Ala, Lys, or Glu.
  • the joining linker is GGS.
  • a joining linker has the sequence (Gly) n where n is any number from 1 to 100, for example: (Gly)s (SEQ ID NO: 34) and (Gly)e (SEQ ID NO: 35).
  • the joining linker is one or more of GGGSE (SEQ ID NO: 47), GSESG (SEO ID NO: 48), GSEGS (SEQ ID NO: 49), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 50), and a joining linker of randomly placed G, S, and E every 4 amino acid intervals.
  • a heterologous chimeric protein used in a method of the present disclosure comprises an extracellular domain (ECD) of SIRPa(CD172a), one joining linker preceding an Fc domain, a second joining linker following the Fc domain, and an ECD of CD40L
  • the chimeric protein may comprise the following structure:
  • SIRPo(CD1723)ECD Joining Linker 1 - Fc Domain - Joining Linker 2 - CD40LECD.
  • a heterologous chimeric protein used in a method of the present disclosure comprises a modular linker as shown in Table 2:
  • a linker may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %,
  • the linker may be flexible, including without limitation highly flexible. In embodiments, the linker may be rigid, including without limitation a rigid alpha helix. Characteristics of illustrative joining linkers is shown below in Table 3:
  • the linker may be functional.
  • the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the chimeric protein used in a method of the present disclosure.
  • the linker may function to target the chimeric protein to a particular cell type or location.
  • a heterologous chimeric protein used in a method of the present disclosure comprises only one joining linkers.
  • a heterologous chimeric protein used in a method of the present disclosure lacks joining linkers.
  • the linker is a synthetic linker such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • a heterologous chimeric protein has a first domain which is sterically capable of binding its ligand/receptor and/or the second domain which is sterically capable of binding its ligand/receptor.
  • first domain which is sterically capable of binding its ligand/receptor
  • second domain which is sterically capable of binding its ligand/receptor.
  • an amino acid sequence may be added to one or more extracellular domains and/or to the linker to provide the slack needed to avoid steric hindrance.
  • Any amino acid sequence that provides slack may be added.
  • the added amino acid sequence comprises the sequence (Gly)n where n is any number from 1 to 100. Additional examples of addable amino acid sequence include the joining linkers described in Table 1 and Table 3.
  • a polyethylene glycol (PEG) linker may be added between an extracellular domain and a linker to provide the slack needed to avoid steric hindrance. Such PEG linkers are well known in the art.
  • a heterologous chimeric protein comprises a first domain comprising a portion of SIRPa(CD172a), a second domain comprising a portion of CD40L, and a linker.
  • the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence.
  • the linker comprises at least about one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH2-CH3 Fc domain.
  • the linker comprises a hinge- CH2-CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4.
  • the linker comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3.
  • a heterologous chimeric protein used in a method of the present disclosure comprises the extracellular domain of SIRPa(CD172a) (or a variant thereof), a linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of CD40L (or a variant thereof), it may be referred to herein as “SIRPa(CD172a)-Fc-CD40L”.
  • a SIRPa(CD172a)-Fc-CD40L chimeric protein of the present disclosure and/or a heterologous chimeric protein used in methods of the present disclosure has the following amino acid sequence:
  • the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about
  • the heterologous chimeric proteins used in methods of the present disclosure are capable of, and can be used in methods comprising, increasing a ratio of effector T cells to regulatory T cells.
  • Illustrative effector T cells include ICOS + effector T cells; cytotoxic T cells (e.g., op TCR, CD3 + , CD8 + , CD45RO+); CD4 + effector T cells (e.g., ap TCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL 7R/CD127 + ); CD8 + effector T cells (e.g., op TCR, CD3 + , CD8 + , CCR7+, CD62Lhi, IL 7R/CD127 + ); effector memory T cells (e.g., CD62Llow, CD44+, TCR, CD3 + , IL7R/CD127 + , IL-15R + , CCR7low); central memoryT cells (e.g.
  • Illustrative regulatory T cells include ICOS + regulatory T cells, CD4 + CD25 + FOXP3 + regulatory T cells, CD4 + CD25 + regulatory T cells, CD4 + CD25- regulatory T cells, CD4 + CD25high regulatory T cells, TIM-3 + PD-1 + regulatory T cells, lymphocyte activation gene-3 (LAG-3) + regulatory T cells, CTLA-4/CD152 + regulatory T cells, neuropilin-1 (Nrp-1) + regulatory T cells, CCR4 + CCR8 + regulatory T cells, CD62L (L-selectin) + regulatory T cells, CD45RBIow regulatory T cells, CD127low regulatory T cells, LRRC32/GARP+ regulatory T cells, CD39 + regulatory T cells, GITR + regulatory T cells, LAP + regulatory T cells, 1 B11 + regulatory T cells, BTLA + regulatory T cells, type 1 regulatory T cells (Tr1 cells), T helper type 3 (Th3) cells, regulatory cell of natural killer T cell phenotype (NKTregs), CD8 + regulatory T
  • the heterologous chimeric proteins used in methods of the present disclosure (and/or additional agents) disclosed herein include derivatives that are modified, /.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition.
  • derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids.
  • FOLR1- immunoconjugates or anti-FO L R 1 immunoconjugates are referred to herein as “FOLR1- immunoconjugates or anti-FO L R 1 immunoconjugates.”
  • SEQ ID NO: 60 and SEQ ID NO: 61 are also provided herein as SEQ ID NO: 60 and SEQ ID NO: 61 , respectively.
  • Anti-FOLR 1 immunoconjugates contain a cell binding agent linked to a cytotoxin.
  • the cell binding agents can be anti-FOLR1 antibodies or antigen-binding fragments thereof.
  • therapeutically effective anti- FOLR1 antibodies can be found in US Appl. Pub. No. US 2012/0009181 which is herein incorporated by reference.
  • An example of a therapeutically effective anti-FOLR1 antibody is huMovI 9 (M9346A) (comprising the sequences of SEQ ID NO: 62 and SEQ ID NO: 64).
  • the polypeptides of SEQ ID NOs: 62-64 comprise the variable domain of the heavy chain of huMovI 9 (M9346A), the variable domain light chain version 1.00, and the variable domain light chain version 1.60 of huMovI 9, respectively.
  • the huMovI 9 anti-FOLR1 antibody is comprised of a variable domain heavy chain represented by SEQ ID NO: 62 and a variable domain light chain represented by SEQ ID NO: 64 (version 1.60 of huMovI 9).
  • the huMovI 9 (M9346A) antibody is encoded by the plasmids deposited with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, VA 20110 on April 7, 2010, under the terms of the Budapest Treaty and having ATCC deposit nos. PTA-10772 and PTA-10774.
  • ATCC American Type Culture Collection
  • the heavy chain sequence of huMov19 comprises a C-terminal terminal lysine (K) after the last glycine (G) of SEQ ID NO: 72 above.
  • the anti-FOLR1 immunoconjugates have one or more of the following effects: inhibit proliferation of tumor cells, reduce the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, inhibit tumor growth, increase patient survival, trigger cell death of tumor cells, differentiate tumorigenic cells to a non-tumorigenic state, or prevent or reduce metastasis of tumor cells.
  • the anti-FOLR1 immunoconjugate comprises an antibody that has antibodydependent cellular cytotoxicity (ADCC) activity.
  • ADCC antibodydependent cellular cytotoxicity
  • the FOLR1 binding molecule is an antibody or antigen-binding fragment comprising the sequences of SEQ ID NOs: 65-69 and the sequence of SEQ ID NO: 71. In some embodiments, the FOLR1 binding molecule is an antibody or antigen-binding fragment comprising the sequences of SEQ ID NOs: 65-68 and the sequences of SEQ ID NOs: 70 and 71 In some embodiments, the FOLR1 binding molecule is an antibody or antigen-binding fragment thereof comprising the sequences of SEQ ID NOs: 65- 68, 78, 70, and 71.
  • polypeptides that comprise a polypeptide having at least about 90% sequence identity to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64.
  • the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64.
  • the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NO: 62 and/or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NO: 63 or SEQ ID NO: 64.
  • the polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO: 62; and/or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
  • the polypeptide is an antibody and/or the polypeptide specifically binds F0LR1.
  • the polypeptide is a murine, chimeric, or humanized antibody that specifically binds FOLR1.
  • the polypeptide having a certain percentage of sequence identity to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64 differs from SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64 by conservative amino acid substitutions only.
  • the polypeptide comprises a polypeptide having at least about 90%, or at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64, wherein the polypeptide comprise the heavy chain CDR amino acid sequences disclosed in Table 4 and the light chain CDR amino acid sequences disclosed in Table 5.
  • Polypeptides can comprise one of the individual light chains or heavy chains described herein. Antibodies and polypeptides can also comprise both a light chain and a heavy chain.
  • polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides comprising an antibody, or fragment thereof, against a human FOLR1.
  • polypeptides and analogs can be further modified to contain additional chemical moieties not normally part of the protein.
  • Those derivatized moieties can improve the solubility, the biological half-life or absorption of the protein.
  • the moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co., Easton, PA (2000).
  • Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. Patent Publication No. 2008/0312425, 2008/0177048, and 2009/0187005, each of which is hereby incorporated by reference herein in its entirety.
  • the drug or prodrug can, for example, be linked to the anti-FOLR1 antibody or fragment thereof through a disulfide bond.
  • the linker molecule or crosslinking agent comprises a reactive chemical group that can react with the anti-FOLR1 antibody or fragment thereof.
  • the reactive chemical groups for reaction with the cellbinding agent can be /V-succinimidyl esters and N-sulfosuccinimidyl esters.
  • the linker molecule comprises a reactive chemical group, which can be a dithiopyridyl group that can react with the drug to form a disulfide bond.
  • Linker molecules include, for example, /V-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) (see US Publication No. 20090274713).
  • the antibody or cell binding agent can be modified with crosslinking reagents and the antibody or cell binding agent containing free or protected thiol groups thus derived is then reacted with a disulfide- or thiol-containing maytansinoid to produce conjugates.
  • the conjugates can be purified by chromatography, including but not limited to HPLC, sizeexclusion, adsorption, ion exchange and affinity capture, dialysis or tangential flow filtration.
  • the anti-FOLR1 antibody is linked to cytotoxic drugs via disulfide bonds and a polyethylene glycol spacer in enhancing the potency, solubility or the efficacy of the immunoconjugate.
  • cytotoxic drugs via disulfide bonds and a polyethylene glycol spacer in enhancing the potency, solubility or the efficacy of the immunoconjugate.
  • cleavable hydrophilic linkers are described in W02009/0134976. The additional benefit of this linker design is the desired high monomer ratio and the minimal aggregation of the antibodydrug conjugate.
  • the linker is a linker containing at least about one charged group as described, for example, in U.S. Patent Publication No. 2012/0282282, the contents of which are entirely incorporated herein by reference.
  • the charged or pro-charged cross-linkers are those containing sulfonate, phosphate, carboxyl or quaternary amine substituents that significantly increase the solubility of the modified cell-binding agent and the cell-binding agent-drug conjugates, especially for monoclonal antibody-drug conjugates with 2 to 20 drugs/antibody linked. Conjugates prepared from linkers containing a pro-charged moiety would produce one or more charged moieties after the conjugate is metabolized in a cell.
  • the linker is selected from the group consisting of: N-succinimidyl 4-(2-pyridyldithio)-2- sulfopentanoate (sulfo-SPP) and N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB).
  • drug load refers to the number of drug molecules (e.g., a maytansinoid) that can be attached to a cell binding agent (e.g., an anti-FOLR1 antibody or fragment thereof).
  • the number of drug molecules that can be attached to a cell binding agent can average from about 2 to about 8 (e.g., 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1).
  • N2’-deacetyl-N2’-(3-mercapto-1 -oxopropyl)-maytansine (DM1) and N2’-deacetyl-N2’- (4-mercapto-4-methyl-1 -oxopentyl) maytansine (DM4) can be used.
  • the maytansinoid (e.g., DM4) is linked (e.g., by sulfo-SPDB) to the anti- FOLR1 antibody or antigen-binding fragment thereof via a lysine residue of the antibody or antigen-binding fragment thereof.
  • 1-10 maytansinoids (e.g., DM4) are linked (e.g., by sulfo-SPDB) to the anti-FOLR1 antibody or antigen-binding fragment thereof via 1-10 lysine residues of the antibody or antigen-binding fragment thereof.
  • 2-8 maytansinoids e.g., DM4 are linked (e.g., by sulfo-SPDB) to the anti-FOLR1 antibody or antigen-binding fragment thereof via 2-8 lysine residues of the antibody or antigen-binding fragment thereof.
  • 2-5 maytansinoids e.g., DM4 are linked (e.g., by sulfo-SPDB) to the anti-FOLR1 antibody or antigen-binding fragment thereof via 2-5 lysine residues of the antibody or antigen-binding fragment thereof.
  • 3-4 maytansinoids e.g., DM4 are linked (e.g., by sulfo-SPDB) to the anti-FOLR1 antibody or antigen-binding fragment thereof via 3- 4 lysine residues of the antibody or antigen-binding fragment thereof.
  • an immunoconjugate comprises 1 maytansinoid per antibody. In another aspect, an immunoconjugate comprises 2 maytansinoids per antibody. In another aspect, an immunoconjugate comprises 3 maytansinoids per antibody. In another aspect, an immunoconjugate comprises 4 maytansinoids per antibody. In another aspect, an immunoconjugate comprises 5 maytansinoids per antibody. In another aspect, an immunoconjugate comprises 6 maytansinoids per antibody. In another aspect, an immunoconjugate comprises 7 maytansinoids per antibody. In another aspect, an immunoconjugate comprises 8 maytansinoids per antibody.
  • an immunoconjugate (e.g., an immunoconjugate comprising the linker sulfo-SPDB and the maytansinoid DM4) comprises about 1 to about 8 maytansinoids per antibody. In another aspect, an immunoconjugate (e.g., an immunoconjugate comprising the linker sulfo-SPDB and the maytansinoid DM4) comprises about 2 to about 7 maytansinoids per antibody. In another aspect, an immunoconjugate (e.g., an immunoconjugate comprising the linker sulfo-SPDB and the maytansinoid DM4) comprises about 2 to about 6 maytansinoids per antibody.
  • an immunoconjugate (e.g., an immunoconjugate comprising the linker sulfo-SPDB and the maytansinoid DM4) comprises about 2 to about 5 maytansinoids per antibody. In another aspect, an immunoconjugate (e.g., an immunoconjugate comprising the linker sulfo-SPDB and the maytansinoid DM4) comprises about 3 to about 5 maytansinoids per antibody. In another aspect, an immunoconjugate (e.g., an immunoconjugate comprising the linker sulfo-SPDB and the maytansinoid DM4) comprises about 3 to about 4 maytansinoids per antibody.
  • a composition comprising immunoconjugates has an average of about 2 to about 8 (e.g., 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1) drug molecules (e.g., maytansinoids) attached per antibody.
  • drug molecules e.g., maytansinoids
  • a composition comprising immunoconjugates has an average of about 1 to about 8 drug molecules (e.g., maytansinoids) per antibody. In one aspect, a composition comprising immunoconjugates has an average of about 2 to about 7 drug molecules (e.g., maytansinoids) per antibody. In one aspect, a composition comprising immunoconjugates has an average of about 2 to about 6 drug molecules (e.g., maytansinoids) per antibody. In one aspect, a composition comprising immunoconjugates has an average of about 2 to about 5 drug molecules (e.g., maytansinoids) per antibody.
  • a composition comprising immunoconjugates has an average of about 3 to about 5 drug molecules (e.g., maytansinoids) per antibody. In one aspect, a composition comprising immunoconjugates has an average of about 3 to about 4 drug molecules (e.g., maytansinoids) per antibody.
  • a composition comprising immunoconjugates has an average of about 2 ⁇ 0.5, about 3 ⁇ 0.5, about 4 ⁇ 0.5, about 5 ⁇ 0.5, about 6 ⁇ 0.5, about 7 ⁇ 0.5, or about 8 ⁇ 0.5 drug molecules (e.g., maytansinoids) attached per antibody. In one aspect, a composition comprising immunoconjugates has an average of about 3.5 ⁇ 0.5 drug molecules (e.g., maytansinoids) per antibody.
  • the anti-FOLR1 antibody or fragment thereof can be modified by reacting a bifunctional crosslinking reagent with the anti-FOLR1 antibody or fragment thereof, thereby resulting in the covalent attachment of a linker molecule to the anti-FOLR1 antibody or fragment thereof.
  • a “bifunctional crosslinking reagent” is any chemical moiety that covalently links a cell-binding agent to a drug, such as the drugs described herein.
  • a portion of the linking moiety is provided by the drug.
  • the drug comprises a linking moiety that is part of a larger linker molecule that is used to join the cell-binding agent to the drug.
  • the side chain at the C-3 hydroxyl group of maytansine is modified to have a free sulfhydryl group (SH).
  • This thiolated form of maytansine can react with a modified cell-binding agent to form a conjugate. Therefore, the final linker is assembled from two components, one of which is provided by the crosslinking reagent, while the other is provided by the side chain from DM1 or DM4.
  • the drug molecules can also be linked to the antibody molecules through an intermediary carrier molecule such as serum albumin.
  • the expression “linked to a cell-binding agent” or “linked to an anti-FOLR1 antibody or fragment” refers to the conjugate molecule comprising at least about one drug derivative bound to a cellbinding agent, anti-FOLR1 antibody, or fragment via a suitable linking group, or a precursor thereof.
  • exemplary linking groups are SPDB or sulfo-SPDB.
  • cytotoxic agents useful in the present invention are maytansinoids and maytansinoid analogs.
  • suitable maytansinoids include esters of maytansinol and maytansinol analogs. Included are any drugs that inhibit microtubule formation and that are highly toxic to mammalian cells, as are maytansinol and maytansinol analogs.
  • suitable maytansinol esters include those having a modified aromatic ring and those having modifications at other positions.
  • Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331 ,598; 4,361 ,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371 ,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497 and 7,473,796.
  • the immunoconjugates of the invention utilize the thiol-containing maytansinoid (DM1), formally termed /V 2 -deacetyl-A/ 2 -(3-mercapto-1 - oxopropyl )-maytansine, as the cytotoxic agent.
  • DM1 is represented by the following structural formula (I):
  • the conjugates of the present invention utilize the thiol-containing maytansinoid W 2 '- deacetyl-/ ⁇ / 2 '(4-methy1-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) as the cytotoxic agent.
  • DM4 is represented by the following structural formula (II):
  • A/ 2 -deacetyl- A/- 2 ’(4-mercapto-1-oxopentyl)-maytansine (termed DM3), represented by the following structural formula (III):
  • positions on maytansinoids can serve as the position to chemically link the linking moiety.
  • the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy and the C-20 position having a hydroxy group are all expected to be useful.
  • the C-3 position serves as the position to chemically link the linking moiety
  • the C-3 position of maytansinol serves as the position to chemically link the linking moiety.
  • M+ is H.
  • a solution of an antibody in aqueous buffer can be incubated with a molar excess of maytansinoids having a disulfide moiety that bears a reactive group.
  • the reaction mixture can be quenched by addition of excess amine (such as ethanolamine, taurine, etc.).
  • the maytansinoid-antibody conjugate can then be purified by gel filtration.
  • the number of maytansinoid molecules bound per antibody molecule can be determined by measuring spectrophotometrically the ratio of the absorbance at 252 nm and 280 nm.
  • the average number of maytansinoid molecules/antibody can be, for example, 1-10 or 2-5.
  • the average number of maytansinoid molecules/antibody can be, for example about 3 to about 4.
  • the average number of maytansinoid molecules/antibody can be about 3.5 or 3.5 +/- 0.5.
  • Conjugates of antibodies with maytansinoid or other drugs can be evaluated for their ability to suppress proliferation of various unwanted cell lines in vitro.
  • cell lines such as the human lymphoma cell line Daudi and the human lymphoma cell line Ramos, can easily be used for the assessment of cytotoxicity of these compounds.
  • Cells to be evaluated can be exposed to the compounds for 4 to 5 days and the surviving fractions of cells measured in direct assays by known methods. IC50 values can then be calculated from the results of the assays.
  • the immunoconjugates can, according to some embodiments described herein, be internalized into cells.
  • the immunoconjugate therefore, can exert a therapeutic effect when it is taken up by, or internalized, by an FOLR1 -expressing cell.
  • the immunoconjugate comprises an antibody, antibody fragment, or polypeptide, linked to a cytotoxic agent by a cleavable linker, and the cytotoxic agent is cleaved from the antibody, antibody fragment, or polypeptide, wherein it is internalized by an FOLR1- expressing cell.
  • the immunoconjugates are capable of reducing tumor volume.
  • treatment with an immunoconjugate results in a %T/C value that is less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%.
  • the immunoconjugates can reduce tumor size in an OVCAR-3, IGROV-1 , and/or OV-90 xenograft model.
  • the immunoconjugates are capable of inhibiting metastases.
  • the methods comprise steps of administering to a subject in need thereof (either simultaneously or sequentially) a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SI RPa(CD172a), (b) a second domain comprising a portion of the extracellular domain of CD40L, and (c) a linker linking the first domain and the second domain; and administering to the subject a second pharmaceutical composition comprising an immunoconjugate of any of the embodiments disclosed herein.
  • a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SI RPa(CD172a), (b) a second domain comprising a portion of the extracellular domain of CD40L, and (c) a linker linking the first domain and the second domain; and administering to the subject a second pharmaceutical composition comprising an immunoconjugate of any of the embodiments disclosed herein.
  • Cancer refers to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system (e.g., virus-infected cells).
  • the cancer may be a primary cancer or a metastatic cancer.
  • the primary cancer may be an area of cancer cells at ovary, peritoneum, or fallopian tube that becomes clinically detectable, and may be a primary tumor.
  • the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part.
  • the metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis.
  • the cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body.
  • the cancer may be due to a process such as lymphatic or hematogeneous spread.
  • the cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor.
  • the cancer may be this new tumor, which may be a metastatic (or secondary) tumor.
  • the cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor.
  • the cells of the tumor may be like those in the original tumor.
  • the secondary tumor while present in the liver, is made up of abnormal ovarian, peritoneal, or fallopian tube cells, not of abnormal liver cells.
  • the tumor in the liver may thus be a metastatic ovarian, peritoneal, or fallopian tube cancer, not liver cancer.
  • ovarian, peritoneal, or fallopian tube cancers of the present disclosure including, but are not limited to, epithelial ovarian cancer (e.g., high-grade serous ovarian cancer (HGSOC), serous carcinomas (high and low grade), including primary peritoneal and/or fallopian tube clear cell carcinoma (CCC), endometrioid carcinoma, mucinous carcinoma, primary peritoneal carcinoma (PPC), fallopian tube cancer (FTC), low-grade serous ovarian carcinoma (LGSOC), squamous cell carcinoma (SCC), borderline epithelial ovarian tumors, and borderline endometrioid ovarian tumors.
  • epithelial ovarian cancer e.g., high-grade serous ovarian cancer (HGSOC), serous carcinomas (high and low grade
  • CCC primary peritoneal and/or fallopian tube clear cell carcinoma
  • endometrioid carcinoma mucinous carcinoma
  • PPC primary peritoneal carcinoma
  • Cancers that can be treated by the methods provided herein include EOC such as primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer and endometrial cancer. Endometrial cancer can also be treated by the methods provided herein.
  • the cancer can be a primary or metastatic cancer.
  • cancers include ovarian epithelial ovarian cancer, ovarian primary peritoneal cancer, or ovarian fallopian tube cancer.
  • the subject has previously untreated ovarian cancer.
  • the subject has previously treated ovarian cancer (e.g., previously treated with a platinum compound, a taxane, bevacizumab, a PARP inhibitor, or a combination thereof).
  • the subject has platinum sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer.
  • the subject has platinum resistant recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer.
  • the cancer is ovarian, peritoneal, or fallopian tube cancer.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a first-line therapy e.g., a second-line therapy, a third-line therapy, or a fourth or later- line therapy.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • the cancer is endometrial cancer.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a first-line therapy e.g., a second- line therapy, a third-line therapy, or a fourth or later-line therapy.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • an endometrial cancer as an adjuvant therapy, neoadjuvant therapy, or maintenance therapy.
  • the cancer is serous endometrial cancer.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a serous endometrial cancer as a first-line therapy, a second-line therapy, a third-line therapy, or a fourth or later-line therapy.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a serous endometrial cancer as an adjuvant therapy, neoadjuvant therapy, or maintenance therapy.
  • the cancer is endometrioid endometrial cancer.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • an endometrioid endometrial cancer as a first-line therapy, a second-line therapy, a third-line therapy, or a fourth or later-line therapy.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • an endometrioid endometrial cancer as an adjuvant therapy, neoadjuvant therapy, or maintenance therapy.
  • the EOC is primary ovarian cancer. In certain embodiments, the EOC is a primary ovarian cancer, a primary fallopian tube cancer or a primary peritoneal cancer. In certain embodiments, the EOC is platinum resistant, relapsed, or refractory or platinum sensitive.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • an EOC e.g., an EOC that is platinum resistant, relapsed, or refractory as a first-line therapy, a second-line therapy, a third-line therapy, or a fourth or later-line therapy.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • an EOC e.g., an EOC that is platinum resistant, relapsed, or refractory as an adjuvant therapy, neoadjuvant therapy, or a maintenance therapy.
  • the cancer is peritoneal cancer.
  • the peritoneal cancer is primary peritoneal cancer.
  • the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) can be administered to a primary peritoneal cancer as a first-line therapy, a second-line therapy, a third-line therapy, or a fourth or later-line therapy.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a primary peritoneal cancer as an adjuvant therapy, a neoadjuvant therapy, or a maintenance therapy.
  • the cancer is platinum refractory. In certain embodiments, the cancer is primary platinum refractory.
  • the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) can be administered to a platinum refractory cancer or a primary platinum refractory cancer as a first-line therapy, a second-line therapy, a third-line therapy, or a fourth or greater-line therapy.
  • an anti- FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a platinum refractory cancer or a primary platinum refractory cancer as an adjuvant therapy, a neoadjuvant therapy, or a maintenance therapy.
  • the cancer is platinum sensitive.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a platinum sensitive cancer as a first-line therapy, a second- line therapy, a third-line therapy, or a fourth or later-line therapy.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a platinum sensitive cancer as an adjuvant therapy, a neoadjuvant therapy, or a maintenance therapy.
  • the cancer is a metastatic or advanced cancer.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a metastatic or advanced cancer as a first-line therapy, a second-line therapy, a third-line therapy, or a fourth or greater-line therapy.
  • an anti- FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a metastatic or advanced cancer as an adjuvant therapy, a neoadjuvant therapy, or a maintenance therapy.
  • Administration of the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) as a "second-line" therapy includes administration wherein the first-line therapy was, for example, administration of a single agent, administration of a combination of agents, surgery, radiation, or a combination thereof.
  • Administration of the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) as a "third-line" therapy includes administration wherein the first-line therapy was, for example, administration of a single agent, administration of a combination of agents, surgery, radiation, or a combination thereof and wherein the second-line therapy was, for example, administration of a single agent, administration of a combination of agents, surgery, radiation, or a combination thereof.
  • administration of the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) as a "third-line" therapy includes for example, administration following a first-line therapy that was administration of a single agent, and a second-line therapy that was administration of a combination of agents.
  • Administration of the combination of an anti-FOLR1 immunoconjugate e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) as a "third-line" therapy also includes, for example, administration following a first-line therapy that was administration of a combination of agents, and a second-line therapy that was administration of a single agent.
  • Administration of the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein also includes, for example, administration following a first-line therapy that was administration of a combination of agents, and a second-line therapy that was administration of a combination of agents.
  • Administration of the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) as a "third-line" therapy also includes, for example, administration following a first- line therapy that was administration of a combination of agents and a surgery, and a second-line therapy that was administration of a combination of agents.
  • a patient receiving administration of the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) has had 1, 2, 3, 4, or more lines of therapy.
  • a patient receiving administration of the combination of an anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) has had no more than 1 , 2, 3, or 4 lines of therapy.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a score is described herein without limitation. Other scoring systems besides the examples provided herein may be utilized according to the implementations disclosed. As applied herein, the description of a score as “moderate” or “medium,” for example, only applies in the context of the scoring system utilized. For example, a sample scored according to heterogenous/homogenous scoring system may be described as “moderate,” but not “medium FRa” according to the H score system. The term “moderate” is not applied in the context of the H scoring system. A sample may be evaluated by more than one scoring system, and there may be overlap in the labeling of that sample. For example, a sample described as “moderate” according to the heterogenous/homogenous scoring system may also be separately scored as “medium” according to the H score system.
  • Wet tissue, tumor blocks, and unstained slides may be obtained. In some instances, unstained, serially-cut, 4-5 microns thick sections may be placed on Superfrost® PLUS slides and obtained from study sites. If a paraffin block is received, six (6) slides may be prepared. If either a paraffin block or a wet tissue (which is then processed into a paraffin block for retrospective study) is received, three (3) slides may be prepared. Specimens may be automatically processed for staining once all accessioning and quality checks have been completed. Slides may be stained with FOLR1 negative and FOLR1 positive markers. In the event that one of the FOLR1 markers fails, a repeat test may be performed.
  • the repeat test may test for the positive and negative markers and may use different available positive and negative FOLR1 markers.
  • a negative marker control for each specimen may be evaluated for acceptable signal/noise ratio and background staining.
  • the negative control may be scored and an assessment of accept or reject can be made.
  • the positive biomarker may be assessed for evaluability based on tissue and cell viability, morphology, and the presence of discernable background staining.
  • H-scores Immunological detection (by immunohistochemistry) of FOLR1 can be scored using H-scores.
  • the percentage of cells staining at each intensity in all relevant cellular compartments e.g., membrane and cytoplasm
  • H-scores combine staining intensity scores (e.g., a score of 0 to 3, wherein 0 represents no staining, and 3 represents strong staining) with the percentage of cells that are positive for membrane staining (/.e., uniformity).
  • An H-score can be calculated as follows:
  • H score [O*(percentage of cells staining at intensity 0)] + [1*(percentage of cells staining at intensity 1)] + [2*(percentage of cells staining at intensity 2)] + [3*(percentage of cells staining at intensity 3)].
  • an H-score can range from 0 (no cell membranes staining) to 300 (all cell membranes staining at intensity 3).
  • Expression of FRa may be defined as “low”, “medium”, or “high” based upon a binned scoring algorithm. “Low expression” refers to a range of at least about 25% of cells to 49% of cells in the sample obtained from the patient having an IHC score of 2 or 3. “Medium expression” refers to a range of at least about 50% of cells to 74% of cells in the sample obtained from the patient having an IHC score of 2 or 3. “High expression” refers to a range of 75% or more cells in the sample obtained from the patient having an IHC score of 2 or 3.
  • Wet tissue, tumor blocks, and unstained slides may be obtained. In some instances, unstained, serially-cut, 4-5 microns thick sections may be placed on Superfrost® PLUS slides and obtained from study sites. If a paraffin block is received, 6 slides may be prepared. If either a paraffin block or a wet tissue (which is then processed into a paraffin block for retrospective study) is received, three (3) slides may be prepared. Specimens may be automatically processed for staining once all accessioning and quality checks have been completed. Slides may be stained with FOLR1 negative and FOLR1 positive markers. In the event that one of the FOLR1 markers fails, a repeat test may be performed. The repeat test may test for the positive and negative markers.
  • a negative marker control for each specimen may be evaluated for acceptable signal/noise ratio and background staining.
  • the negative control may be scored and an assessment of accept or reject can be made.
  • the positive biomarker may be assessed for evaluability based on tissue and cell viability, morphology, and the presence of discernable background staining.
  • the FOLR1 protein expression can also be measured by immunohistochemistry (IHC). The percentage of cells staining in all relevant cellular compartments e.g., membrane and cytoplasm), may be obtained.
  • the FOLR1 biomarker slides may be assessed for tumor cell membrane staining per the scoring algorithm provided in Table 8 below:
  • a third method of cancer scoring is a heterogeneous I homogeneous scoring system.
  • the cancer is a cancer that expresses FOLR1 (polypeptide or nucleic acid).
  • FOLR1 polypeptide or nucleic acid
  • the combination of the anti-FOLR1 immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) is administered to a patient with an increased expression level of FOLR1 , for example, as described in U.S. Published Application No. 2012/0282175 or International Published Application No. WO 2012/135675, both of which are incorporated by reference herein in their entireties.
  • Exemplary antibodies, assays, and kits for the detection of FOLR1 are provided in WO 2014/036495 and WO 2015/031815, both of which are incorporated by reference herein in their entireties.
  • the FOLR1 protein expression has been measured by immunohistochemistry (IHC) and given a staining intensity score and/or a staining uniformity score by comparison to controls (e.g., calibrated controls) exhibiting defined scores (e.g., an intensity score of 3 is given to the test sample if the intensity is comparable to the level 3 calibrated control or an intensity of 2 (moderate) is given to the test sample if the intensity is comparable to the level 2 calibrated control).
  • controls e.g., calibrated controls
  • defined scores e.g., an intensity score of 3 is given to the test sample if the intensity is comparable to the level 3 calibrated control or an intensity of 2 (moderate) is given to the test sample if the intensity is comparable to the level 2 calibrated control.
  • a staining uniformity that is “heterogeneous” or “hetero” (/.e., at least about 25% and less than 75% cells stained).
  • a staining uniformity that is “homogeneous” or “homo” (/.e., at least about 75% cells stained) instead of "focal” (/.e., greater than 0% and less than 25% cells stained) is also indicative of increased FOLR1 expression.
  • the staining intensity and staining uniformity scores can be used alone or in combination (e.g., 2 homo, 2 hetero, 3 homo, 3 hetero, etc.).
  • an increase in FOLR1 expression can be determined by detection of an increase of at least about 2-fold, at least about 3-fold, or at least about 5-fold) relative to control values (e.g., expression level in a tissue or cell from a subject without cancer or with a cancer that does not have elevated FOLR1 values).
  • control values e.g., expression level in a tissue or cell from a subject without cancer or with a cancer that does not have elevated FOLR1 values.
  • the staining uniformity score can be based on the percent of stained cells.
  • the cancer can be a cancer that expresses FOLR1 at a level of 1 hetero or higher by IHC.
  • the cancer can be a cancer that expresses FOLR1 at a level of 2 hetero or higher by IHC.
  • the cancer can be a cancer that expresses FOLR1 at a level of 3 hetero or higher by IHC.
  • the cancer can be a primary ovarian cancer, primary fallopian tube cancer or primary peritoneal cancer that expresses FOLR1 at a level of 2 hetero or higher by IHC.
  • the cancer can be a primary ovarian cancer, primary fallopian tube cancer or primary peritoneal cancer that expresses FOLR1 at a level of 3 hetero or higher by IHC.
  • the cancer also can be an endometrial cancer that expresses FOLR1 at a level of 2 hetero or higher by IHC.
  • a least one cell is present in a sample obtained from a patient has an FOLR1 score of at least about 1 . At least about one cell in sample obtained from a patient can have an FOLR1 score of at least about 2 (moderate). At least about one cell in sample obtained from a patient can have an FOLR1 score of at least about 3.
  • At least about 25% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 1 .
  • at least about 33% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 1.
  • at least about 50% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 1 .
  • at least about 66% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 1 .
  • at least about 75% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 1 .
  • At least about 25% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 2 (“moderate”).
  • at least about 33% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 2 (“moderate”).
  • 25-75% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 2 (“moderate”).
  • at least about 50% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 2 (“moderate”).
  • At least about 66% of the cells in a sample obtained from a patient have a FOLR1 1 HC score of at least about 2 (“moderate”). In some embodiments, at least about 75% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 2 (moderate).
  • at least about 25% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 3.
  • at least about 33% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 3.
  • at least about 50% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 3.
  • At least about 66% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 3. In some embodiments, at least about 75% of the cells in a sample obtained from a patient have a FOLR1 IHC score of at least about 3.
  • membrane FRa expression can be measured by immunohistochemistry (IHC) and given a “staining intensity score” and/or a “staining uniformity score” by comparison to calibrated controls exhibiting defined scores (e.g., an intensity score of 3+ is given to the test sample if the intensity is comparable to the level 3+ calibrated control or an intensity of 2+ is given to the test sample if the intensity is comparable to the level 2+ calibrated control).
  • a score of 0 refers to no staining.
  • a score of 1 + refers to light (brown) staining.
  • a score of 2+ refers to medium (brown) staining, and a score of 3+ refers to dark (brown) staining.
  • Staining uniformity can be expressed as percentage (%) of cells staining at a certain intensity (e.g., 50% of cells staining at intensity of 1+, 2+, or 3+).
  • PS refers to percentage stained.
  • >75% of cells with PS2+ staining intensity indicates that at least 75% of cells in sample have a staining intensity of at least 2+ (i.e., 2+ or 3+).
  • the present disclosure provides a method for treating an epithelial ovarian cancer (EOC) in a subject in need thereof comprising: administering to the subject a first pharmaceutical composition comprising a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SIRPa(CD172a), wherein the portion is capable of binding a SIRPa(CD172a) ligand, (b) a second domain comprising a portion of the extracellular domain of CD40L, wherein the portion is capable of binding a CD40L receptor, and (c) a linker linking the first domain and the second domain; and administering to the subject a second pharmaceutical composition comprising an immunoconjugate, wherein the immunoconjugate comprises a maytansinoid compound and an anti-FOLR1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementary determining region (CDR)1 sequence of SEQ ID NO: 68, a VH CDR2 sequence of S
  • the first pharmaceutical composition and the second pharmaceutical composition are administered contemporaneously. In embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition is administered. In embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition is administered.
  • the dose of the first pharmaceutical composition is less than the dose of the first pharmaceutical composition administered to a second subject who has not undergone or is not undergoing treatment with the second pharmaceutical composition.
  • the dose of the second pharmaceutical composition administered is less than the dose of the second pharmaceutical composition administered to a second subject who has not undergone or is not undergoing treatment with the first pharmaceutical composition.
  • the subject has an increased chance of survival, without gastrointestinal inflammation and weight loss, and/or a reduction in tumor size or cancer prevalence when compared to a subject who has only undergone or is only undergoing treatment with the first pharmaceutical composition. In embodiments, the subject has an increased chance of survival, without gastrointestinal inflammation and weight loss, and/or a reduction in tumor size or cancer prevalence when compared to a subject who has only undergone or is only undergoing treatment with the second pharmaceutical composition.
  • the present disclosure provides a method for treating a cancer in a subject comprising: administering to the subject a pharmaceutical composition comprising a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SIRPo(CD172a), wherein the portion is capable of binding a SI RPa(CD172a) ligand, (b) a second domain comprising a portion of the extracellular domain of CD40L, wherein the portion is capable of binding a CD40L receptor, and (c) a linker linking the first domain and the second domain; wherein the subject has undergone or is undergoing treatment with a second pharmaceutical composition comprising an immunoconjugate, wherein the immunoconjugate comprises a maytansinoid compound and an anti-FOLR1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementary determining region (CDR)1 sequence of SEQ ID NO: 68, a VH CDR2 sequence of SEQ ID NO: 69
  • the present disclosure provides a method for treating a cancer in a subject comprising: administering to the subject a second pharmaceutical composition comprising an immunoconjugate, wherein the immunoconjugate comprises a maytansinoid compound and an anti-FOLR1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementary determining region (CDR)1 sequence of SEQ ID NO: 68, a VH CDR2 sequence of SEQ ID NO: 69, and a VH CDR3 sequence of SEQ ID NO: 71 , and a light chain variable region (VL) CDR1 sequence of SEQ ID NO: 65, a VL CDR2 sequence of SEQ ID NO: 66, and a VL CDR3 sequence of SEQ ID NO: 67; wherein the subject has undergone or is undergoing treatment with a heterologous chimeric protein comprising: (a) a first domain comprising a portion of the extracellular domain of SIRPa(CD172a), wherein the portion is
  • the dose of the pharmaceutical composition provided to the subject is less than the dose of the pharmaceutical composition that is provided to a second subject who has not undergone or is not undergoing treatment with the heterologous chimeric protein.
  • the heterologous chimeric protein comprises a first domain which comprises substantially the entire extracellular domain of SIRPo(CD172a) and/or a second domain which comprises substantially the entire extracellular domain of CD40L.
  • the heterologous chimeric protein comprises: (a) a first domain comprising an extracellular domain of SIRPa(CD172a), (b) a second domain comprising an extracellular domain of CD40L, and (c) a linker comprising a hinge-CH2-CH3 Fc domain.
  • the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence.
  • the linker comprises at least about one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH2-CH3 Fc domain.
  • the linker comprises a hinge-CH2-CH3 Fc domain derived from I gG 1 or I gG4, e.g., human lgG4 or human I gG4.
  • the linker comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  • the first domain comprises an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 57.
  • the second domain comprises an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 58.
  • the heterologous chimeric protein comprises: (a) a first domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 57, (b) a second domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 58, and (c) a linker comprising a hinge-CH2-CH3 Fc domain derived from human lgG4.
  • the heterologous chimeric protein comprises an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 59.
  • the anti-FOLR1 antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 62 and a VL comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 64.
  • the anti-FOLR1 antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 72 and a light chain comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74.
  • the maytansinoid is selected from mertansine (DM1), DM3, and DM4.
  • the maytansinoid is DM4.
  • maytanisonid is linked to the antibody or antigen-binding fragment thereof by a sulfo-SPDB linker.
  • the immunoconjugate comprises 1-10 maytansinoid molecules, 2-8 maytansinoid molecules, 2-5 maytansinoid molecules, or 3-5 maytansinoid molecules, or 3-4 maytansinoid molecules.
  • the immunoconjugate has the following chemical structure:
  • the immunoconjugate comprises 2-5 or 3-4 maytansinoid molecules.
  • the immunoconjugate is administered once every three weeks. In embodiments, the immunoconjugate is administered at a dose in the range of about 1.5 mg/kg to about 4.5 mg/kg adjusted ideal body weight (AIBW), or about 3 mg/kg to about 9 mg/kg AIBW, or about 4.5 mg/kg to about 13.5 mg/kg AIBW, or about 6 mg/kg to about 18 mg/kg AIBW.
  • AIBW adjusted ideal body weight
  • the immunoconjugate is administered at a dose of about 2 mg/kg, or about 3 mg/kg, or about 4 mg/kg, or about 6 mg/kg, or about 8 mg/kg, or about 10 mg/kg, or about 12 mg/kg, or about 14 mg/kg, or about 16 mg/kg, or about 18 mg/kg adjusted ideal body weight (AIBW). In embodiments, the immunoconjugate is administered at a dose of about 3 mg/kg, or about 6 mg/kg adjusted ideal body weight (AIBW).
  • the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 10 mg/kg. In embodiments, the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 0.9 mg/kg, or about 0.5 mg/kg to about 1 .5 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 6 mg/kg, or about 3 mg/kg to about 9 mg/kg, or about 4 mg/kg to about 10 mg/kg. In embodiments, the heterologous chimeric protein is administered at a dose of about 0.3 mg/kg, or about 1 mg/kg, or about 3 mg/kg or about 10 mg/kg.
  • the heterologous chimeric protein is administered at a dose of about 0.3 mg/kg, or about 1 mg/kg, or about 3 mg/kg or about 10 mg/kg), optionally wherein the heterologous chimeric protein is administered once a week or once every two weeks, or once every three weeks (Q3W); and the immunoconjugate is administered at a dose of about 5 mg/kg, or about 6 mg/kg adjusted ideal body weight (AIBW), optionally wherein the immunoconjugate is administered once every three weeks (Q3W).
  • AIBW ideal body weight
  • the heterologous chimeric protein is administered at a dose in the range of about 3 mg/kg to about 10 mg/kg and the immunoconjugate is administered at a dose of about 5 mg/kg or about 6 mg/kg adjusted ideal body weight (AIBW) administered Q3W, wherein the immunoconjugate comprises an average of 3-4 maytansinoid molecules per antibody.
  • AIBW ideal body weight
  • the heterologous chimeric protein is administered once a week or once every two weeks. In embodiments, the heterologous chimeric protein is administered once a week. In embodiments, the heterologous chimeric protein is administered once every two weeks. In embodiments, the heterologous chimeric protein is administered in a biphasic or multiphasic regimen of administration comprising once a week or once every two weeks administrations.
  • the EOC originated in the ovary, fallopian tube, or peritoneum.
  • EOC is a primary ovarian cancer, a primary fallopian tube cancer or a primary peritoneal cancer.
  • the EOC is platinum resistant, relapsed, or refractory.
  • the subject has a histologically confirmed diagnosis of primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer, or high grade serous epithelial ovarian cancer.
  • the subject has received at least about one but no more than two prior systemic lines of anticancer therapy. In embodiments, the subject has received at least about one but no more than three prior systemic lines of anticancer therapy.
  • the subject has received at least about one but no more than five prior systemic lines of anticancer therapy. In embodiments, the subject has received at least about one line of therapy comprising bevacizumab or is medically-ineligible to receive bevacizumab. In embodiments, the subject is relapsed after receiving in a platinum-based therapy. In embodiments, the administration results in a decrease in CA125.
  • the EOC expresses FOLR1 protein.
  • the FOLR1 protein expression has been measured by immunohistochemistry (IHC) in a tumor sample obtained from the subject.
  • IHC immunohistochemistry
  • a staining score of at least about 1 hetero, at least about 1 homo, at least about 2 hetero, at least about 2 homo, or at least about 3 hetero is measured by IHC.
  • at least about 25%, at least about 33%, at least about 50%, at least about 66%, or at least about 75% of cells in the sample obtained from the subject have an IHC score of at least about 2.
  • the subject has been determined to be FRo positive by the analysis of biopsy samples (without limitation, e.g., fresh core needle biopsy samples) that are collected from the subject.
  • the subject has been determined to be FRo positive as determined by membrane staining visible at less than or equal to 10x microscope objective.
  • the subject has been determined to be FRo positive as determined by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • about 25% to about 49% of cells in the sample have an IHC score of at least about 2. In embodiments, about 50% to about 74% of cells in the sample have an IHC score of at least about 2. In embodiments, about 75% to about 100% of cells in the sample have an IHC score of at least about 2.
  • the epithelial ovarian cancer exhibits a detectable level surface expression of FOLR1 protein.
  • the level of surface expression of FOLR1 protein is measured by immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS), RNA sequencing, or a combination thereof.
  • the epithelial ovarian cancer (EOC) exhibits a low FOLR1 protein expression as characterized by IHC score (e.g., having at least about 25% of cells to 49% of cells in the sample obtained from the patient having an IHC score of 2).
  • the epithelial ovarian cancer expresses CD47 and/or calreticulin.
  • the EOC expresses CD47, calreticulin and/or CD40.
  • the administration of the immunoconjugate increases the expression and/or activity of CD47, calreticulin, and/or annexin V in an sample of the EOC compared to a control sample comprising an EOC sample from a subject that is obtained prior to the administration of the immunoconjugate or an EOC sample from a different subject that is not been treated with the immunoconjugate, or a healthy tissue from the subject or from a different subject, optionally wherein the expression and/or activity of CD47, calreticulin, and/or annexin V is measured by RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof, optionally wherein the expression and/or activity of CD47, calreticulin, and/or annexin V is measured by contacting the sample with an agent that: specifically binds CD47, calreticulin, and/or annexin V, optional
  • the immunoconjugate and the heterologous chimeric protein are administered in separate pharmaceutical compositions.
  • the immunoconjugate is administered intravenously or intraperitoneally.
  • the heterologous chimeric protein is administered intravenously.
  • the administration of the heterologous chimeric protein and the immunoconjugate is a first- line therapy. In embodiments, administration is a second-line therapy. In embodiments, the administration of the heterologous chimeric protein and the immunoconjugate is a third-line or later than third line therapy.
  • the subject has previously been treated with a platinum compound, a taxane, bevacizumab, a PARP inhibitor, or a combination thereof.
  • the EOC is primary platinum refractory.
  • the EOC is platinum resistant.
  • the EOC is metastatic or advanced.
  • the administration of the heterologous chimeric protein and the immunoconjugate produces a greater therapeutic benefit than administration of the heterologous chimeric protein alone or the immunoconjugate alone. In embodiments, the administration of the heterologous chimeric protein and the immunoconjugate does not produce more toxicity than the toxicity produced by the administration of the heterologous chimeric protein alone or the immunoconjugate alone.
  • the administration of the heterologous chimeric protein and the immunoconjugate increases or stimulated the level of phagocytosis of EOC cells compared to the level of phagocytosis of EOC cells prior to the administration of the immunoconjugate or an EOC sample from a different subject that is not been treated with the heterologous chimeric protein and the immunoconjugate.
  • the administration of the heterologous chimeric protein and the immunoconjugate does not stimulate apoptosis of macrophages compared to or drugs that macrophage apoptosis (e.g., bisphosphonates).
  • the present disclosure provides a method of treating a patient having epithelial ovarian cancer (EOC) cancer comprising administering to said patient in need thereof a first pharmaceutical composition comprising about 0.3 mg/Kg to about 10 mg/Kg of a heterologous chimeric protein wherein the heterologous chimeric protein comprises a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain is capable of binding a SIRPa(CD172a) ligand and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 57, (b) is a second domain is capable of binding a CD40L receptor and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 58, and (c) is a linker adjoining (a) and (b) and comprising a hinge-CH2-CH3 Fc domain derived from human lg
  • the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 0.9 mg/kg, or about 0.5 mg/kg to about 1 .5 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 6 mg/kg, or about 3 mg/kg to about 9 mg/kg, or about 4 mg/kg to about 10 mg/kg.
  • the immunoconjugate is administered at a dose in the range of about 1 .5 mg/kg to about 4.5 mg/kg adjusted ideal body weight (AIBW), or about 3 mg/kg to about 9 mg/kg AIBW, or about 4.5 mg/kg to about 13.5 mg/kg AIBW, or about 6 mg/kg to about 18 mg/kg AIBW.
  • AIBW adjusted ideal body weight
  • the present disclosure provides a method of treating a patient having epithelial ovarian cancer (EOC) cancer comprising administering to said patient in need thereof a first pharmaceutical composition comprising about 0.3 mg/Kg to about 10 mg/Kg of a heterologous chimeric protein wherein the heterologous chimeric protein comprises a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain is capable of binding a SIRPa(CD172a) ligand and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 57, (b) is a second domain is capable of binding a CD40L receptor and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 58, and (c) is a linker adjoining (a) and (b) and comprising a hinge-CH2-CH3 Fc domain derived from human lg
  • the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 0.9 mg/kg, or about 0.5 mg/kg to about 1 .5 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 6 mg/kg, or about 3 mg/kg to about 9 mg/kg, or about 4 mg/kg to about 10 mg/kg.
  • the immunoconjugate is administered at a dose in the range of about 1 .5 mg/kg to about 4.5 mg/kg adjusted ideal body weight (AIBW), or about 3 mg/kg to about 9 mg/kg AIBW, or about 4.5 mg/kg to about 13.5 mg/kg AIBW, or about 6 mg/kg to about 18 mg/kg AIBW.
  • AIBW adjusted ideal body weight
  • the present disclosure provides a method of treating a patient having epithelial ovarian cancer (EOC) cancer comprising administering to said patient in need thereof a second pharmaceutical composition comprising 6 mg/kg AIBW of an immunoconjugate, wherein the immunoconjugate comprises an antibody linked to the maytansinoid DM4 by a sulfo-SPDB linker, wherein the an antibody comprises (i) a heavy chain comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 72 and (ii) a light chain comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74; wherein the subject has undergone or is undergoing treatment with a first
  • the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 0.9 mg/kg, or about 0.5 mg/kg to about 1.5 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 6 mg/kg, or about 3 mg/kg to about 9 mg/kg, or about 4 mg/kg to about 10 mg/kg.
  • the immunoconjugate is administered at a dose in the range of about 1.5 mg/kg adjusted ideal body weight (AIBW) to about 4.5 mg/kg AIBW, or about 3 mg/kg AIBW to about 9 mg/kg AIBW, or about 4.5 mg/kg AIBW to about 13.5 mg/kg AIBW, or about 6 mg/kg AIBW to about 18 mg/kg AIBW.
  • AIBW adjusted ideal body weight
  • the present disclosure provides a method of treating a patient having epithelial ovarian cancer (EOC) cancer comprising administering to said patient in need thereof a first pharmaceutical composition comprising about 0.3 mg/Kg to about 10 mg/Kg of a heterologous chimeric protein, wherein the heterologous chimeric protein comprises a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising the extracellular domain of SIRPa(CD172a) and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 57, wherein the extracellular domain is capable of binding a SIRPo(CD172a) ligand, (b) is a second domain comprising the extracellular domain of CD40L and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 58, wherein the extracellular domain is capable of binding a CD40L receptor
  • the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 0.9 mg/kg, or about 0.5 mg/kg to about 1 .5 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 6 mg/kg, or about 3 mg/kg to about 9 mg/kg, or about 4 mg/kg to about 10 mg/kg.
  • the immunoconjugate is administered at a dose in the range of about 1 .5 mg/kg to about 4.5 mg/kg AIBW, or about 3 mg/kg to about 9 mg/kg AIBW, or about 4.5 mg/kg to about 13.5 mg/kg AIBW, or about 6 mg/kg to about 18 mg/kg AIBW.
  • the present disclosure provides a method of treating a patient having epithelial ovarian cancer (EOC) cancer comprising administering to said patient in need thereof a first pharmaceutical composition comprising about 0.3 mg/Kg to about 10 mg/Kg of a heterologous chimeric protein, wherein the heterologous chimeric protein comprises a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising the extracellular domain of SIRPo(CD172a) and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 57, wherein the extracellular domain is capable of binding a SIRPo(CD172a) ligand, (b) is a second domain comprising the extracellular domain of CD40L and comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 58, wherein the extracellular domain is capable of binding a CD40L receptor
  • the immunoconjugate is administered at a dose in the range of about 1 .5 mg/kg to about 4.5 mg/kg AIBW, or about 3 mg/kg to about 9 mg/kg AIBW, or about 4.5 mg/kg to about 13.5 mg/kg AIBW, or about 6 mg/kg to about 18 mg/kg AIBW.
  • the present disclosure provides a method of treating a patient having epithelial ovarian cancer (EOC) cancer comprising administering to said patient in need thereof a second pharmaceutical composition comprising 6 mg/kg AIBW of an immunoconjugate, wherein the immunoconjugate comprises an antibody linked to the maytansinoid DM4 by a sulfo-SPDB linker, wherein the an antibody comprises (i) a heavy chain comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of the heavy chain encoded by the plasmid deposited with the American Type Culture Collection (ATCC) as PTA-10772 and (ii) a light chain comprising an amino acid sequence that is at least about 90%, or at least about 93%, at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% identical to the amino acid sequence of the
  • the heterologous chimeric protein is administered at a dose in the range of about 0.3 mg/kg to about 0.9 mg/kg, or about 0.5 mg/kg to about 1.5 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 6 mg/kg, or about 3 mg/kg to about 9 mg/kg, or about 4 mg/kg to about 10 mg/kg.
  • the immunoconjugate is administered at a dose in the range of about 1 .5 mg/kg to about 4.5 mg/kg AIBW, or about 3 mg/kg to about 9 mg/kg AIBW, or about 4.5 mg/kg to about 13.5 mg/kg AIBW, or about 6 mg/kg to about 18 mg/kg AIBW.
  • the first pharmaceutical composition and the second pharmaceutical composition are administered contemporaneously. In embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition is administered. In embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition is administered.
  • the linker comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  • the heterologous chimeric protein comprises an amino acid sequence that is at least about 95%, or at least about 96%, or at least about 98%, or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 59.
  • the immunoconjugate comprises 1-10, 2-8, 2-5, or 3-4 maytansinoids. In embodiments, the immunoconjugate comprises 2-5 or 3-4 maytansinoids. In embodiments, the immunoconjugate has the following chemical structure: wherein “Ab” represents the anti-FOLR 1 antibody or antigen binding fragment thereof.
  • the epithelial ovarian cancer (EOC) exhibits a detectable level surface expression of FOLR1 protein. In embodiments, the level of surface expression of FOLR1 protein is measured by immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS), RNA sequencing, or a combination thereof.
  • the epithelial ovarian cancer exhibits a low FOLR1 protein expression as characterized by IHC score (e.g., having at least about 25% of cells to 49% of cells in the sample obtained from the patient having an IHC score of 2).
  • the epithelial ovarian cancer expresses CD47 and/or calreticulin.
  • the EOC has originated in the ovary cancer, fallopian tube, or peritoneum cancer.
  • EOC is a primary ovarian cancer, a primary fallopian tube cancer or a primary peritoneal cancer.
  • the EOC is platinum resistant, relapsed, or refractory.
  • the patient has a histologically confirmed diagnosis of primary ovarian cancer, primary fallopian tube cancer, primary peritoneal cancer, or high grade serous epithelial ovarian cancer.
  • the patient has received at least about one but no more than three prior systemic lines of anticancer therapy.
  • the patient has received at least about one line of therapy comprising bevacizumab or is medically-ineligible to receive bevacizumab.
  • the combination of the anti-FOLR1 immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) is useful for inhibiting tumor growth.
  • the combination of the anti-FOLR1 immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL- 172154) is useful for reducing or preventing metastasis.
  • the combination of the anti- FOLR1 immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) is useful for reducing tumor volume.
  • treatment with a combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein results in a reduction in tumor size, mass, or volume.
  • the combination of a FOLR1 immunoconjugate e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein is capable of inhibiting metastases.
  • the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) can reduce the tumorigenicity of a tumor.
  • the methods of use can be in vivo methods.
  • the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein produces a synergistic effect.
  • administration of the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) does not produce more toxicity than administration of the anti-PD-1 antibody or antigen-binding fragment thereof.
  • administration of the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) does not produce more toxicity than administration of the anti-FOLR1 immunoconjugate.
  • administration of the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein does not produce more toxicity than administration of either the anti-FOLR1 immunoconjugate or the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154).
  • Each of the above aspects can further include monitoring the subject for recurrence of the cancer. Monitoring can be accomplished, for example, by evaluating progression free survival (PFS), overall survival (OS), objective response rate (ORR) complete response (CR), partial response (PR).
  • PFS progression free survival
  • OS overall survival
  • ORR objective response rate
  • CR complete response
  • PR partial response
  • the PFS is evaluated after initiation of treatment. In some embodiments, PFS is extended by about 3-6 months, compared to a control. In one embodiment, the PFS is extended by about 3 months with the treatment regimen combining a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) compared to a control.
  • a FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • the PFS is extended by at least about 4 months with the treatment regimen combining a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) compared to a control.
  • the PFS is extended by about 5 months with the treatment regimen combining a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) compared to a control.
  • the PFS is extended by about 6 months with the treatment regimen combining a F0LR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) compared to a control.
  • a F0LR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • the total PFS time after treatment with the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL- 172154) is about 3 months to 1 year.
  • the total PFS time is about 3 months.
  • the total PFS time is about 4 months.
  • the total PFS time is about 5 months.
  • the total PFS time is about 6 months.
  • the total PFS time is about 7 months.
  • the total PFS time is about 8 months.
  • the total PFS time is about 9 months.
  • the total PFS time is about 10 months.
  • the total PFS time is about 11 months.
  • the total PFS time is about 1 year.
  • the total PFS time is about 6 to 9 months.
  • the total PFS time is about 6 to 8 months.
  • the objective response rate is the proportion of patients achieving a complete response partial response or stable disease (CR, PR or SD).
  • the treatment provided herein achieves an ORR of at least about 25%. In one embodiment, the treatment provided herein achieves an ORR of about 30%. In one embodiment, the treatment provided herein achieves an ORR of about 35%. In one embodiment, the treatment provided herein achieves an ORR of about 40%. In one embodiment, the treatment provided herein achieves an ORR of about 45%. In one embodiment, the treatment provided herein achieves an ORR of about 50%. In one embodiment, the treatment provided herein achieves an ORR of 25-50%.
  • treatment with FOLR1 immunoconjugate e.g., IMGN853 and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) increase PFS and ORR.
  • the total PFS can be about 3 months to about 1 year and the ORR can be at least about 25%.
  • the total PFS can be about 3 months to about 1 year and the ORR can be about 25-50%.
  • the total PFS can be about 9 months and the ORR can be at least about 25%.
  • the total PFS can be about 9 months and the ORR can be about 25-50%.
  • a steroid can be administered in addition to the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154).
  • a FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • the administration of the steroid in addition to the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and an the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) results in a reduction of headaches as compared to administration of only the combination of a F0LR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154).
  • the steroid can be administered at the same time as the immunoconjugate, prior to the administration of the immunoconjugate, and/or after the administration of the immunoconjugate. In some embodiments, the steroid is administered within about a week, about five days, about three days, about two days, or about one day or 24 hours prior to the administration of the immunoconjugate. In some embodiments, the steroid is administered within one day of the administration of the immunoconjugate. In some embodiments, the steroid is administered about 30 minutes prior to the administration of the immunoconjugate. In some embodiments, the steroid is administered multiple times. In some embodiments, the steroid is administered about one day prior to the administration of the immunoconjugate and on the same day as the administration of the immunoconjugate.
  • the steroid can be administered via any number of ways, including for example, topical, pulmonary, oral, parenteral, or intracranial administration.
  • the administration is oral.
  • the administration is intravenous.
  • the administration is both oral and intravenous.
  • acetaminophen/paracetamol, dexamethasone, and/or diphenhydramine can be administered prior to (e.g., 30 minutes prior to) administration of the immunoconjugate (e.g., IMGN853).
  • the immunoconjugate e.g., IMGN853
  • 325-650 mg acetaminophen/paracetamol (orally or intravenously), 10 mg dexamethasone (intravenously), and/or 25-50 mg diphenhydramine (orally or intravenously) can be administered prior to (e.g., 30 minutes prior to) administration of the immunoconjugate (e.g., IMGN853).
  • a steroid is administered in an eye drop (e.g., a corticosteroid eye drop, including, but not limited to: cortisol, glucocorticoid, dexamethasone, cortisone, prednisolone, fluocinolone, difluprednate, loteprednol, fluoromethoIone, triamcinolone, rimexolone).
  • the eye drops are preservative-free, lubricating eye drops.
  • the steroid in the eye drop is dexamethasone.
  • a lubricating eye drop can be administered in addition to the ophthalmic steroid, which is administered to reduce the ocular toxicity associated with the administration of an anti-FOLR1 immunoconjugate (e.g., IMGN853).
  • the lubricating eye drop can be administered to reduce dry eye.
  • the lubricating eye drops are preservative-free, lubricating eye drops.
  • the lubricating eye drops are not administered on the same day as the ophthalmic steroid (e.g., administered after an ophthalmic steroid). In other embodiments, the lubricating eye drops are administered on the same day as the ophthalmic steroid.
  • Another analgesic or other medication to prevent or treat headaches can also be administered in addition to the combination of a FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154).
  • a FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL- 172154
  • the analgesic can be administered prior to, at the same time, or after the administration of the immunoconjugate and can be via any appropriate administration route. In some embodiments, the analgesic is administered orally.
  • Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • IMGN853 can be administered at a particular dose and/or at particular timing intervals.
  • Administration of anti-FOLR1 immunoconjugates can be, for example, intravenous or intraperitoneal.
  • Dosing regiments for anti-FORL1 immunoconjugates are provided, for example, in WO 2014/186403, WO 2015/054400, and WO 2015/149018, each of which is herein incorporated by reference in its entirety.
  • an anti- FOLR1 immunoconjugate e.g., I MGN853
  • I MGN853 can be administered at a dose of about 6 mg/kg, wherein the kilograms of body weight are based on adjusted ideal body weight (AIBW).
  • AIBW adjusted ideal body weight
  • an anti-FOLR1 immunoconjugate (e.g., IMGN853) is administered every three weeks.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853 is administered every three weeks at a dose of about 6 mg/kg AIBW.
  • An anti-FOLR1 immunoconjugate (e.g., IMGN853) can be administered at a dose of about 5 mg/kg AIBW. In some embodiments, an anti-FOLR1 immunoconjugate (e.g., IMGN853) is administered every three weeks at a dose of about 5 mg/kg AIBW.
  • the heterologous chimeric protein of any of the embodiments disclosed herein can be administered at a particular dose and/or at particular timing intervals.
  • Administration of a heterologous chimeric protein of any of the embodiments disclosed herein can be, for example, intravenous.
  • a heterologous chimeric protein of any of the embodiments disclosed herein is administered every three weeks (Q3W). In some embodiments, a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) is administered at a dose of about 200 mg. In some embodiments, a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) is administered every three weeks at a dose of about 200 mg.
  • a heterologous chimeric protein of any of the embodiments disclosed herein is administered every three weeks administered at a dose of about 200 mg and an anti-FOLR1 immunoconjugate (e.g., IMGN853) is administered every three weeks at a dose of about 6 mg/kg AIBW.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein is administered every three weeks administered at a dose of about 200 mg and an anti-FOLR1 immunoconjugate (e.g., IMGN853) is administered every three weeks at a dose of about 5 mg/kg AIBW.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • the immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) are administered simultaneously.
  • the anti- FOLR1 immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) are administered in separate pharmaceutical compositions.
  • the anti-FOLR1 immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) are administered in the same pharmaceutical composition.
  • the anti-FOLR1 immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) are administered sequentially.
  • the anti-FOLR1 immunoconjugate (e.g., IMGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) are administered sequentially, and the immunoconjugate is administered before anti-FOLR1 immunoconjugate (e.g., IMGN853).
  • Routes of administration include, for example: intradermal, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local.
  • Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.
  • the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure are administered in the tumor microenvironment (e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor) or lymph node and/or targeted to the tumor microenvironment or lymph node.
  • the tumor microenvironment e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vascul
  • heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure allows for a dual effect that provides less side effects than are seen in conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ).
  • heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure reduce or prevent commonly observed immune- related adverse events that affect various tissues and organs including the skin, the gastrointestinal tract, the kidneys, peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and the endocrine system; such as hypophysitis, colitis, hepatitis, pneumonitis, rash, and rheumatic disease.
  • the present local administration obviate adverse event seen with standard systemic administration, e.g., IV infusions, as are used with conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ).
  • Dosage forms suitable for parenteral administration include, for example, solutions, suspensions, dispersions, emulsions, and the like.
  • sterile solid compositions e.g., lyophilized composition
  • sterile injectable medium immediately before use.
  • They may contain, for example, suspending or dispersing agents known in the art.
  • the dosage of the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) disclosed herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject’s general health, and the administering physician’s discretion.
  • heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure, disclosed herein can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof.
  • an additional agent e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72
  • the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure and an additional agent(s) are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.
  • the present disclosure relates to the co-administration of the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure which induces an innate immune response and another antibody directed to immune checkpoint molecules; and/or chimeric protein used in methods of the present disclosure which induces an adaptive immune response.
  • the heterologous chimeric proteins of any embodiment disclosed herein used in methods of the present disclosure which induces an innate immune response may be administered before, concurrently with, or subsequent to administration of the second pharmaceutical composition comprising the immunoconjugate of any embodiment disclosed herein.
  • heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure may be administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.
  • first pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed used in methods of the present disclosure which induces an innate immune response and the second pharmaceutical composition comprising herein the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure which induces an adaptive response are administered 1 week apart, or administered on alternate weeks (/.e., administration of the second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure inducing an innate immune response is followed 1 week later with administration of the second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure which induces an adaptive immune response and so forth).
  • the dosage of a second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) disclosed herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used.
  • the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.
  • the dosage may be about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day.
  • the dosage of any agent disclosed herein may be about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day, or about 200 to about 1,200 mg per day (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 ,000 mg, about 1 ,100 mg, about 1 ,200 mg per day).
  • administration of the second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) disclosed herein is by parenteral injection at a dosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mg to about 10 mg per treatment, or about 0.5 mg to about 5 mg per treatment, or about 200 to about 1 ,200 mg per treatment (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 ,000 mg, about 1,100 mg, about 1 ,200 mg per treatment).
  • a suitable dosage of the second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight or about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1 .1 mg/kg, about 1 .2 mg/kg, about 1 .
  • delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat ef al., in Liposomes in Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
  • a liposome see Langer, 1990, Science 249:1527-1533; Treat ef al., in Liposomes in Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
  • a second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) disclosed herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety.
  • Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions.
  • Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 ; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71 :105).
  • a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533 may be used.
  • Administration of a second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) disclosed herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject.
  • the dosage regimen utilizing a second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) disclosed herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the present disclosure employed.
  • the second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) disclosed herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily.
  • a second pharmaceutical composition comprising the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure (and/or additional agents) disclosed herein can be administered continuously rather than intermittently throughout the dosage regimen.
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154) used in a method of the present disclosure may be a recombinant fusion protein, e.g., a single polypeptide having the extracellular domains disclosed herein.
  • the chimeric protein is translated as a single unit in a prokaryotic cell, a eukaryotic cell, or a cell-free expression system.
  • a heterologous chimeric protein of any of the embodiments disclosed herein is recombinant protein comprising multiple polypeptides, e.g., multiple extracellular domains disclosed herein, that are combined (via covalent or non-covalent bonding) to yield a single unit, e.g., in vitro (e.g., with one or more synthetic linkers disclosed herein).
  • a heterologous chimeric protein of any of the embodiments disclosed herein is chemically synthesized as one polypeptide or each domain may be chemically synthesized separately and then combined.
  • a portion of the chimeric protein is translated and a portion is chemically synthesized.
  • Constructs could be produced by cloning of the nucleic acids encoding the three fragments (the extracellular domain of SIRPa(CD172a), followed by a linker sequence, followed by the extracellular domain of CD40L into a vector (plasmid, viral or other) wherein the amino terminus of the complete sequence corresponded to the ‘left’ side of the molecule containing the extracellular domain of the SIRPa(CD172a) and the carboxy terminus of the complete sequence corresponded to the ‘right’ side of the molecule containing the extracellular domain of CD40L.
  • a vector plasmid, viral or other
  • a construct would comprise three nucleic acids such that the translated chimeric protein produced would have the desired configuration, e.g., a dual inward-facing chimeric protein. Accordingly, in embodiments, the chimeric proteins used in methods of the present disclosure are engineered as such.
  • a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) used in a method of the present disclosure may be encoded by a nucleic acid cloned into an expression vector.
  • the expression vector comprises DNA or RNA.
  • the expression vector is a mammalian expression vector.
  • Prokaryotic vectors include constructs based on E. coll sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512- 538).
  • Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 and APL.
  • Non-limiting examples of prokaryotic expression vectors may include the Agt vector series such as Agt11 (Huynh et a/., in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp.
  • Prokaryotic host-vector systems cannot perform much of the post-translational processing of mammalian cells, however. Thus, eukaryotic host- vector systems may be particularly useful.
  • a variety of regulatory regions can be used for expression of the chimeric proteins in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used.
  • CMV cytomegalovirus
  • RSV-LTR Rous sarcoma virus long terminal repeat
  • Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the 0- interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the chimeric proteins in recombinant host cells.
  • promoters associated with the metallothionein II gene mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the 0- interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75).
  • expression vectors comprise a nucleic acid encoding the chimeric proteins, or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell.
  • the expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent-encoding nucleic acid such that the blocking and/or stimulating agent is produced in a human cell transformed with the expression vector.
  • a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL- 172154) used in a method of the present disclosure is producible in a mammalian host cell as a secretable and fully functional single polypeptide chain.
  • Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid.
  • An expression control region of an expression vector of the present disclosure is capable of expressing operably linked encoding nucleic acid in a human cell.
  • the cell is a tumor cell.
  • the cell is a non-tumor cell.
  • the expression control region confers regulatable expression to an operably linked nucleic acid.
  • a signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region.
  • Such expression control regions that increase expression in response to a signal are often referred to as inducible.
  • Such expression control regions that decrease expression in response to a signal are often referred to as repressible.
  • the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.
  • the present disclosure contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue.
  • inducible promoters capable of effecting high level of expression transiently in response to a cue.
  • a cell transformed with an expression vector for the chimeric protein (and/or additional agents) comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue.
  • Illustrative inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound.
  • the chimeric protein is expressed by a chimeric antigen receptor containing cell or an in vitro expanded tumor infiltrating lymphocyte, under the control of a promoter which is sensitive to antigen recognition by the cell, and leads to local secretion of the chimeric protein in response to tumor antigen recognition.
  • a promoter which is sensitive to antigen recognition by the cell, and leads to local secretion of the chimeric protein in response to tumor antigen recognition.
  • Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function.
  • the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).
  • operable linkage refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner.
  • the relationship is such that the control element modulates expression of the nucleic acid.
  • an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (/.e., “upstream”).
  • Expression control regions can also be located at the 3’ end of the transcribed sequence (/.e., “downstream”) or within the transcript (e.g., in an intron).
  • Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid).
  • a specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence.
  • Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.
  • a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA.
  • a promoter will have a transcription-initiating region, which is usually placed proximal to the 5' end of the coding sequence, and, typically, a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
  • promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • the 3’ terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation.
  • transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs.
  • nucleic acids there is a variety of techniques available for introducing nucleic acids into viable cells.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc.
  • liposomes For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction.
  • a targeting agent such as an antibody or ligand specific for a tumor cell surface membrane protein.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).
  • gene delivery agents such as, e.g., integration sequences can also be employed.
  • Numerous integration sequences are known in the art (see, e.g., Nunes-Duby ef al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacterio!., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol.
  • transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).
  • direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the chimeric fusion proteins including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.
  • the expression vectors for the expression of the chimeric proteins (and/or additional agents) are viral vectors.
  • Many viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol., 21 : 1 17, 122, 2003.
  • Illustrative viral vectors include those selected from antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and a viruses, though other viral vectors may also be used.
  • LV antiviruses
  • RV retroviruses
  • AV adenoviruses
  • AAV adeno-associated viruses
  • viral vectors that do not integrate into the host genome are suitable for use, such as a viruses and adenoviruses.
  • viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV).
  • VEE Venezuelan equine encephalitis
  • SFV Semliki Forest virus
  • viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and antiviruses.
  • the present disclosure provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the present disclosure.
  • Expression vectors can be introduced into host cells for producing the chimeric proteins used in methods of the present disclosure.
  • Cells may be cultured in vitro or genetically engineered, for example.
  • Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler in “Gene Transfer and Expression: A Laboratory Manual,” 1990, New York, Freeman & Co.).
  • monkey kidney cell lines transformed by SV40 e.g., COS-7, ATCC CRL 1651
  • human embryonic kidney lines e.g., 293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59
  • baby hamster kidney cells e.g., BHK, ATCC CCL 10
  • Chinese hamster ovary-cells-DHFR e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216
  • DG44 CHO cells CHO-K1 cells, mouse sertoli cells (Mather, Biol Reprod 1980, 23:243-251)
  • mouse fibroblast cells e.g., NIH-3T3
  • monkey kidney cells e.g., CV1 ATCC CCL 70
  • African green monkey kidney cells e.g., VERO-76, ATCC CRL-1587
  • human cervical carcinoma cells e.g.
  • Illustrative cancer cell types for expressing the chimeric proteins disclosed herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, EG7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.
  • Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection (ATCC), or from commercial suppliers.
  • Cells that can be used for production of the chimeric proteins used in methods of the present disclosure in vitro, ex vivo, and/or in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, chimeric antigen receptor expressing T cells, tumor infiltrating lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, and fetal liver.
  • Fc-containing macromolecules such as monoclonal antibodies
  • Fc-containing macromolecules are produced by human embryonic kidney (HEK) cells (or variants thereof) or Chinese Hamster Ovary (CHO) cells (or variants thereof) or in some cases by bacterial or synthetic methods.
  • HEK human embryonic kidney
  • CHO Chinese Hamster Ovary
  • the Fc containing macromolecules that are secreted by HEK or CHO cells are purified through binding to Protein A columns and subsequently ‘polished’ using various methods.
  • purified Fc containing macromolecules are stored in liquid form for some period of time, frozen for extended periods of time or in some cases lyophilized.
  • production of the chimeric proteins contemplated herein may have unique characteristics as compared to traditional Fc containing macromolecules.
  • the chimeric proteins may be purified using specific chromatography resins or using chromatography methods that do not depend upon Protein A capture.
  • the chimeric proteins may be purified in an oligomeric state, or in multiple oligomeric states, and enriched for a specific oligomeric state using specific methods. Without being bound by theory, these methods could include treatment with specific buffers including specified salt concentrations, pH and additive compositions. In other examples, such methods could include treatments that favor one oligomeric state over another.
  • the chimeric proteins obtained herein may be additionally ‘polished’ using methods that are specified in the art.
  • the chimeric proteins are highly stable and able to tolerate a wide range of pH exposure (between pH 3-12), are able to tolerate a large number of freeze/thaw stresses (greater than 3 freeze/thaw cycles) and are able to tolerate extended incubation at high temperatures (longer than 2 weeks at 40 degrees C). In embodiments, the chimeric proteins are shown to remain intact, without evidence of degradation, deamidation, etc. under such stress conditions. Subjects and/or Animals
  • the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.
  • the subject and/or animal is a non-mammal, such, for example, a zebrafish.
  • the subject and/or animal may comprise fluorescently tagged cells (with e.g., GFP).
  • the subject and/or animal is a transgenic animal, which comprises a fluorescent cell.
  • the subject and/or animal is a human.
  • the human is a pediatric human.
  • the human is an adult human.
  • the human is a geriatric human.
  • the human may be referred to as a patient.
  • the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
  • the subject is a non-human animal, and therefore the present disclosure pertains to veterinary use.
  • the non-human animal is a household pet.
  • the non-human animal is a livestock animal.
  • anti-FOLR1 immunoconjugates e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • the cancer can be a primary ovarian cancer, primary fallopian tube cancer or primary peritoneal cancer. In some instances, the cancer can be endometrial cancer.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853 and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) are contained within the same pharmaceutical composition.
  • an anti-FOLR1 immunoconjugate e.g., IMGN853
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a kit comprises an anti-FOLR1 immunoconjugate (e.g., IMGN853) and instructions to administer the anti-FOLR1 immunoconjugate (e.g., IMGN853) and a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154).
  • a kit comprises a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) and instructions to administer the heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) and an anti-FOLR1 immunoconjugate (e.g., IMGN853).
  • the pharmaceutical compositions provided herein comprise an anti-FOLR1 immunoconjugate (e.g., IMGN853) and/or a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) and a pharmaceutically acceptable vehicle.
  • the pharmaceutical compositions further comprise a preservative. These pharmaceutical compositions find use in inhibiting tumor growth and treating cancer in human patients.
  • compositions for use as provided herein can be administered in any number of ways, for example, by parenteral administration including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion.
  • the pharmaceutical composition is formulated for intravenous (i.v.) administration.
  • the pharmaceutical composition is formulated for intraperitoneal (i.p.) administration.
  • the anti-FOLR1 immunoconjugate (I MGN853) and the heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • kits can simplify the administration of the pharmaceutical compositions and/or chimeric proteins disclosed herein, the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein
  • kits of the present disclosure comprises the heterologous chimeric proteins of any embodiment disclosed herein and/or the immunoconjugate of any embodiment disclosed herein used in methods of the present disclosure and/or pharmaceutical composition disclosed herein in unit dosage form.
  • the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent disclosed herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.
  • the kit can further comprise a label or printed instructions instructing the use of any agent disclosed herein.
  • the kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location.
  • the kit can also further comprise one or more additional agent disclosed herein.
  • the kit comprises a container containing an effective amount of a composition of the present disclosure and an effective amount of another composition, such those disclosed herein.
  • aspects of the present disclosure include use of a heterologous chimeric protein of any of the embodiments disclosed herein (e.g., SL-172154) as disclosed herein in the manufacture of a medicament, e.g., a medicament for treatment of cancer.
  • a heterologous chimeric protein of any of the embodiments disclosed herein e.g., SL-172154
  • a medicament for treatment of cancer e.g., a medicament for treatment of cancer.
  • the examples herein are provided to illustrate advantages and benefits of the present disclosure and to further assist a person of ordinary skill in the art with using the combination of the SIRPa-Fc-CD40L chimeric protein (SL-172154) and Mirvetuximab for treating ovarian cancer.
  • the examples herein are also presented in order to more fully illustrate the preferred aspects of the present disclosure.
  • the examples should in no way be construed as limiting the scope of the present disclosure, as defined by the appended claims.
  • the examples can include or incorporate any of the variations, aspects or embodiments of the present disclosure described above.
  • the variations, aspects or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present disclosure.
  • Ovarian carcinoma cell line KB was grown in the presence of vehicle only control or 0.2 pg/ml, 1 pg/ml, 10 pg/ml, or 50 pg/ml Mirvetuximab for 1 , 3, 6 or 24 hours and calreticulin, CD40, Mirvetuximab-bound folate receptor a (FRa), annexin V (a marker of apoptosis) and CD47 were analyzed by flow cytometry.
  • FIG. 2A shows the surface expression of calreticulin, CD40 and Mirvetuximab-bound folate receptor a (FRa) after incubation for 1 , 3, 6 or 24 hours.
  • FIG. 2B shows the surface expression of annexin V (a marker of apoptosis) and CD47 after incubation for 1 , 3, 6 or 24 hours.
  • IGROV-1 human ovarian carcinoma cells were grown in the presence of vehicle only control or 0.2 pg/ml, 1 pg/ml, 10 pg/ml, or 50 pg/ml Mirvetuximab for 1, 3, 6 or 24 hours and calreticulin, CD40, Mirvetuximab-bound folate receptor a (FRa), annexin V (a marker of apoptosis) and CD47 were analyzed by flow cytometry.
  • FIG. 3A shows the surface expression of calreticulin, CD40 and Mirvetuximab-bound folate receptor a (FRa) after incubation for 1 , 3, 6 or 24 hours.
  • FIG. 3B shows the surface expression of annexin V (a marker of apoptosis) and CD47 after incubation for 1 , 3, 6 or 24 hours.
  • FIG. 4A shows the surface expression of calreticulin, CD40 and Mirvetuximab-bound folate receptor a (FRa) after incubation for 1 , 3, 6 or 24 hours.
  • FIG. 4B shows the surface expression of annexin V (a marker of apoptosis) and CD47 after incubation for 1 , 3, 6 or 24 hours.
  • FIG. 5A shows the surface expression of calreticulin, CD40 and Mirvetuximab-bound folate receptor a (FRa) after incubation for 1 , 3, 6 or 24 hours.
  • FIG. 5B shows the surface expression of annexin V (a marker of apoptosis) and CD47 after incubation for 1 , 3, 6 or 24 hours.
  • Ovarian cancer cell lines KB, IGROV-1 , OV90 and SKOV-3 were cultured in the presence of vehicle only control or 0.2 pg/ml, 1 pg/ml, 10 pg/ml, or 50 pg/ml Mirvetuximab for 3 or 24 hours and CD47, CD40, calreticulin, annexin V (a marker of apoptosis), and Mirvetuximab-bound folate receptor a (FRa) were analyzed by flow cytometry.
  • annexin V a marker of apoptosis
  • ovarian cancer cell lines KB, IGROV-1 , OV90 and SKOV-3 after 3 hours (FIG. 6G) or 24 hours (FIG. 6H) in the presence of Mirvetuximab was plotted.
  • the Mirvetuximab receptor (FRa) occupancy in ovarian cancer cell lines KB, IGROV-1, OV90 and SKOV-3 after 3 hours (FIG. 6I) or 24 hours (FIG. 6J) in the presence of Mirvetuximab was plotted.
  • FIG. 8 shows a summary of flow phenotyping.
  • CD47 and CD40 surface expression were assessed by flow cytometry on the cell lines above at baseline.
  • Calreticulin (CALR), Annxin V, and FRa were assessed before and after incubation of tumor cells with Mirvetuximab.
  • FRa receptor occupancy Mirvetuximab was used as the cell surface detection reagent.
  • each of the tested ovarian cancer cell line showed expression of CD40.
  • three of the four tested ovarian cancer cell lines showed expression of Calreticulin (CALR, FIG. 8).
  • three of the four tested ovarian cancer cell lines showed expression of folate receptor alpha (FRa, FIG.
  • Ovarian cancer cell lines KB, IGROV-1, OV90, SKOV-3 and MES-0V3 were cultured in the presence of vehicle only control or 0.2 pg/ml, 1 pg/ml, 10 pg/ml, or 50 pg/ml Mirvetuximab for 3 hr and Mirvetuximab- bound folate receptor a (FRa) was analyzed by flow cytometry.
  • KB and IGROV-1 cells showed high level of the FRa receptor occupancy, indicating a high level of folate receptor a expression in these cells.
  • OV90, SKOV-3 and MES-OV3 cells showed low level of the FRa receptor occupancy, indicating a low level of folate receptor a expression in these cells (FIG. 6K).
  • Ovarian cancer cell lines KB, IGROV-1, OV90, SKOV-3 and MES-OV3 were cultured and stained with anti- CD47 or anti-calreticulin (CALR) antibodies, and were analyzed by flow cytometry.
  • IGROV-1 , OV90, and SKOV-3 cells showed low level of the calreticulin expression.
  • KB cells showed an intermediate level of calreticulin expression (FIG. 6L).
  • MES-OV3 cells showed a high level of calreticulin expression (FIG. 6L).
  • IGROV-1, OV90, and SKOV-3 cells showed low level of the CD47 expression.
  • KB and MES-OV3 cells showed an intermediate level of CD47 expression.
  • these cells provided various variations in the expression of folate receptor a, calreticulin and CD47.
  • KB cells showed high level of folate receptor a and CD47 expression but intermediate level of calreticulin expression.
  • MES-OV3 cells expressed low levels of folate receptor a expression but high levels of calreticulin and CD47 expression.
  • IGROV-1 cells expressed high levels of folate receptor a but low levels of calreticulin and CD47.
  • OV-90 and SKOV3 cells expressed low levels of each of folate receptor a, calreticulin and CD47 expression.
  • the baseline surface expression of CD47, CALR, and folate receptor a was accessed in a battery of commercially available ovarian cancer cell lines. Specifically, the following cells were grown and stained with anti-CD47 or anti-calreticulin antibodies: HPV-related endocervical adenocarcinoma cells KB, ovarian endometrioid adenocarcinoma cells IGROV-1 , ovarian adenocarcinoma cells OV-90 (metastatic site ascites), ovarian serous cystadenocarcinoma cells SK-OV-3 (metastatic site ascites), ovarian serous cystadenocarcinoma cells MES-OV3 (metastatic site ascites), ovarian clear cell adenocarcinoma cells TOV-21 G, high-grade ovarian serous adenocarcinoma cells HEYT30, ovarian endometrioid adenocarcinoma cells TOV-112
  • FIG. 7A these cells exhibited varying levels of expression of CD47.
  • FIG. 7B these cells exhibited varying levels of expression of calreticulin.
  • HPV-related endocervical adenocarcinoma cells KB, IGROV-1, OV-90, SK-OV-3, MES-OV3, TOV-21G, HEYT30, TOV-112D, A2780, and 59M cells were grown in the presence of 10 g/ml Mirvetuximab and analyzed by flow cytometry for receptor occupancy.
  • FIG. 7C these cells exhibited varying levels of expression of folate receptor a.
  • FIG. 7A to FIG. 7C these cells provided numerous variations in the expression of folate receptor a, calreticulin and CD47.
  • the KB ovarian cancer cells showed a significant increase in apoptosis when grown in the presence of 1 pg/ml Mirvetuximab compared to KB cells cultured in the presence of vehicle alone control (p ⁇ 0.001). Similarly, the KB ovarian cancer cells showed an increase in apoptosis when grown in the presence of 10 pg/ml Mirvetuximab compared to KB cells cultured in the presence of vehicle alone control (FIG. 9A).
  • the OV90 ovarian cancer cells showed a significant increase in apoptosis when grown in the presence of 10 pg/ml Mirvetuximab compared to OV90 cells cultured in the presence of vehicle alone control (p ⁇ 0.01). Similarly, the OV90 ovarian cancer cells showed an increase in apoptosis when grown in the presence of 1 pg/ml Mirvetuximab compared to OV90 cells cultured in the presence of vehicle alone control (FIG. 9B).
  • the KB, IGROV-1 , OV90 and MES-OV-3 ovarian cancer cells were labeled the IncuCyte phRodo Red cell labeling kit and co-cultured with human PBMC-derived M1 macrophages and treated with (1) vehicle alone control, (2) 50 pg/ml of the SIRPa-Fc-CD40L chimeric protein (SL-172154), (3) 10 pg/ml Mirvetuximab, or (4) 10 pg/ml Mirvetuximab and 50 pg/ml of the SIRPa-Fc-CD40L chimeric protein (SL-172154) and incubated at 37°C in the presence of 5% CO2 for 2 hours.
  • vehicle alone control (2) 50 pg/ml of the SIRPa-Fc-CD40L chimeric protein (SL-172154), (3) 10 pg/ml Mirvetuximab, or (4) 10 pg/ml Mirvetuximab
  • phagocytosis was determined by an increase in red fluorescent intensity which occurs when the phRodo Red labeled tumor cell is internalized into the acidic macrophage phagosome.
  • a phagocytosis index was calculated by setting the maximum phagocytosis value to 1 , and then normalizing all other replicates accordingly. The phagocytosis index was plotted for the indicated treatments.
  • the KB ovarian cancer cells that were treated with vehicle alone control were phagocytized by the human macrophages at a background level (indicated by a dotted line in FIG. 10A).
  • the treatment with Mirvetuximab alone (p ⁇ 0.001) or the SIRPa-Fc-CD40L chimeric protein (SL-172154) alone (p ⁇ 0.05) resulted in a significant increase in the level of phagocytosis compared to the vehicle only-treated KB ovarian cancer cells (FIG. 10A).
  • FIG. 10A the treatment with Mirvetuximab alone (p ⁇ 0.001) or the SIRPa-Fc-CD40L chimeric protein (SL-172154) alone (p ⁇ 0.05)
  • the IGROV1 ovarian cancer cells that were treated with vehicle alone control were phagocytized by the human macrophages at a background level (indicated by a dotted line in FIG. 10B).
  • the OV90 ovarian cancer cells that were treated with vehicle alone control were phagocytized by the human macrophages at a background level (indicated by a dotted line in FIG. 10C).
  • the treatment with Mirvetuximab alone (p ⁇ 0.05) or the SIRPa-Fc-CD40L chimeric protein (SL-172154) alone resulted in a significant increase in the level of phagocytosis compared to the vehicle only-treated OV90 ovarian cancer cells (FIG. 10C).
  • FIG. 10C the OV90 ovarian cancer cells that were treated with vehicle alone control were phagocytized by the human macrophages at a background level (indicated by a dotted line in FIG. 10C).
  • the treatment with Mirvetuximab alone (p ⁇ 0.05) or the SIRPa-Fc-CD40L chimeric protein (SL-172154) alone resulted in a significant increase in the level of phagocytos
  • OV-90 cells expressed low levels of each of folate receptor a, calreticulin and CD47 expression.
  • the data in FIG. 10C show, inter alia, that the combination of SL-172154 with Mirvetuximab especially broadens the phagocytic activity in CD47
  • the MES-OV3 ovarian cancer cells that were treated with vehicle alone control were phagocytized by the human macrophages at a background level (indicated by a dotted line in FIG. 10D).
  • the treatment with the SIRPa-Fc-CD40L chimeric protein (SL-172154) alone resulted in an increase in the level of phagocytosis compared to the vehicle only-treated MES-OV3 ovarian cancer cells (FIG. 10D).
  • the treatment with Mirvetuximab alone showed a small increase in the level of phagocytosis compared to the vehicle only-treated MES-OV3 ovarian cancer cells (FIG. 10D).
  • FIG. 10D shows that shows that were treated with vehicle alone control.
  • PBMC-derived monocytes were differentiated into M1 macrophages. Macrophages were then treated with low/high doses of Mirvetuximab, SL-172154, or the combination of Mirvetuximab and SL-172154. After 3 or 24 hours, apoptosis was assessed in the macrophages using annexin V staining and flow cytometry.
  • FIG. 11 A there was minimal induction of apoptosis, as indicated by annexin V staining, in PBMC- derived M1 macrophages, if any, when cultured in the presence of low concentrations of Mirvetuximab (1 pg/ml), the SIRPa-Fc-CD40L chimeric protein (SL-172154, 10 pg/ml), or a combination of the two.
  • Example 5 An Open-Label, Phase 1b Study of SL-172154 (SIRPa-Fc-CD40L) Administered with Mirvetuximab Soravtansine in Subjects with Platinum-Resistant Ovarian Cancers
  • a Phase 1 b clinical trial for evaluating the SIRPo-Fc-CD40L chimeric protein (SL-172154) as combination therapy with Mirvetuximab in patients with ovarian cancer will be carried out.
  • Objectives for this study include evaluation of safety, tolerability, pharmacokinetics, anti-tumor activity, pharmacodynamic effects of SL- 172154 and Mirvetuximab, and to select the recommended Phase 2 dose (RP2D) for SL-172154 administered with Mirvetuximab in subjects with platinum-resistant ovarian, primary peritoneal, or fallopian tube cancer.
  • Study design is shown in FIG. 12.
  • this will be an open-label, multicenter, Phase 1 b human clinical trial designed to evaluate the safety, PK, PD effects, and preliminary anti-tumor activity of SL-172154 administered as add-on therapy to background treatment with Mirvetuximab in subjects with platinum-resistant ovarian, primary peritoneal, or fallopian tube cancers.
  • the study will consist of dose escalation followed by dose expansion (see Study Schema).
  • Subjects with histologically confirmed epithelial ovarian cancer or primary peritoneal or fallopian tube cancer of high-grade histology who are platinum-resistant will be eligible.
  • Subjects having a tumor that is positive for FRa expression as defined by central testing using the Ventana FOLR1 Assay will be eligible.
  • FRa expression in tumor samples is analyzed using the Ventana FOLR1 Assay, an immunohistochemical assay developed to detect FRa in cut slide specimens of formalin-fixed paraffin-embedded (FFPE) epithelial ovarian cancer tissue stained on the BenchMark ULTRA automated staining instrument using the Ventana OptiView DAB IHC Detection Kit.
  • Biopsy samples without limitation, e.g., fresh core needle biopsy samples
  • tumor tissue blocks are collected from the subjects are used for analysis of FRo expression prior to enrollment.
  • fresh cut slides from available blocks e.g., FFPE slides
  • a scoring method based on membrane stain intensity level of 2 or greater is used for patient selection.
  • the method is referred to herein as PS2+.
  • the PS2+ scoring method requires assessment of the percentage of tumor cells with moderate (2) and/or strong (3) membrane staining compared to the total number of viable tumor cells. For example, patients with tumors having >25% of viable tumor cells exhibiting level 2 and/or 3 membrane staining intensity are considered positive for FRa expression and are eligibility for the study. Eligible patients include subgroups with high (PS2+ >75%), medium (PS2+ >50% and ⁇ 75%), or low (PS2+ >25% and ⁇ 50%) tumor FRa expression in subjects with platinum-resistant ovarian, primary peritoneal, or fallopian tube cancer.
  • subjects may be eligible if at least about 25% of cells in the sample obtained from the subject have an IHC score of at least about level 2 membrane staining intensity.
  • Subjects may be eligible as having low tumor FRa expression if about 25% to about 49% of cells in the sample have an IHC score of at least about level 2 membrane staining intensity.
  • Subjects may be eligible as having medium tumor FRa expression if about 50% to about 74% of cells in the sample have an IHC score of at least about level 2 membrane staining intensity.
  • Subjects may be eligible as having high tumor FRa expression if about 75% to about 100% of cells in the sample have an IHC score of at least about level 2 membrane staining intensity.
  • Subjects that are platinum-resistant disease as defined as follows will be eligible: • Subjects who have only had 1 line of platinum-based therapy and have received at least about 4 cycles of platinum and have had a response (complete response/remission [CR] or partial response/remission [PR]) and then progressed between >3 months and ⁇ 6 months after the date of the last dose of platinum.
  • CR complete response/remission
  • PR partial response/remission
  • Adjuvant ⁇ neoadjuvant is considered 1 line of therapy
  • Maintenance therapy e.g., bevacizumab, PARP inhibitors
  • will be considered part of the preceding line of therapy /.e., not counted independently.
  • Hormonal therapy will be counted as a separate line of therapy unless it was given as maintenance.
  • the study will initially enroll subjects to the SL-172154 + MIRV escalation cohorts.
  • the SL-172154 starting dose of 3 mg/kg is supported by safety data from the SL03-OHD-101 study in which 3 subjects receiving 1 mg/kg and 5 subjects receiving 3 mg/kg of SL-172154 once-weekly have cleared the DLT evaluation period with no DLTs reported.
  • the initial SL-172154 dose escalation cohorts in Study SL03-OHD-105 will be SL-172154 (3.0 mg/kg, IV; Days 8 and 15 of each 21-day cycle) administered with Mirvetuximab (6mg/kg AIBW (adjusted ideal body weight), IV; Day 1 of each 21-day cycle).
  • subjects will be enrolled in sequential cohorts of approximately 3 subjects and evaluated for dose-limiting toxicity (DLT) during the first cycle of therapy (/.e., DLT evaluation period). Treatment will be administered in 21-day cycles until disease progression, unacceptable toxicity, or withdrawal of consent. Dose escalation of SL-172154 with each of the combination regimens will continue until a safe dose of SL-172154 when administered with Mirvetuximab is identified.
  • the dose escalation cohorts will utilize the modified Toxicity Probability Interval (mTPI-2) design [Guo, 2017] with target DLT rate of 30% for the maximum tolerated dose (MTD).
  • an expansion cohort will enroll approximately 70 subjects whose tumor is FRa positive (including subjects from dose escalation at the same dose level) to further evaluate the safety and efficacy of the study treatment. All subjects with FRa positive tumors will be enrolled and retrospectively binned by FRa expression subgroups (high, medium, or low) in the study analysis. The same patient population will be enrolled in both the dose escalation and dose expansion portion of the study.
  • Approximately 12 subjects will be enrolled in dose escalation cohorts and approximately 64 subjects (including subjects from dose escalation at the same dose level) will be enrolled in the expansion cohorts.
  • the planned sample sizes may be revised if additional dose levels are evaluated or if more subjects (/.e., subjects available for dosing beyond the number required in a cohort) are enrolled than anticipated.
  • the actual number of subjects to be enrolled for each combination dose escalation will depend upon the number of dose levels evaluated and the number of DLTs observed for each dose level and related dose escalation/stay/de-escalation decision.
  • SL-172154 as combination therapy with Mirvetuximab will be safe and tolerable and will show anti-tumor activity in patients with ovarian cancer.

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Abstract

La présente divulgation concerne, entre autres, des combinaisons des protéines chimériques hétérologues qui comprennent une partie de SIRPo et une partie de CD40L qui sont jointes par un lieur et un immunoconjugué, lesquelles ont une utilisation dans des méthodes de traitement du cancer de l'ovaire, telles que des immunothérapies pour le cancer de l'ovaire.
PCT/US2023/071006 2022-07-26 2023-07-26 Polythérapie pour le traitement du cancer de l'ovaire Ceased WO2024026340A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110097334A1 (en) * 2007-10-02 2011-04-28 Universitat Zu Koln Novel Marker Genes for Regulatory T Cells from Human Blood
US20120244171A1 (en) * 2011-02-15 2012-09-27 Immunogen, Inc. Cytotoxic benzodiazepine derivatives
US20180333503A1 (en) * 2017-05-16 2018-11-22 Immunogen, Inc. Anti-folr1 immunoconjugates and anti-pd-1 antibody combinations
WO2020176718A1 (fr) * 2019-02-28 2020-09-03 Shattuck Labs, Inc. Polythérapies
US20200362029A1 (en) * 2019-04-29 2020-11-19 ImmnoGen, Inc. Biparatopic fr-alpha antibodies and immunoconjugates
US20220119519A1 (en) * 2018-08-08 2022-04-21 Orionis Biosciences, Inc. Sirp1a targeted chimeric proteins and uses thereof
WO2023086929A1 (fr) * 2021-11-12 2023-05-19 Shattuck Labs, Inc. Protéines chimériques à base de sirp1a et cd40l

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110097334A1 (en) * 2007-10-02 2011-04-28 Universitat Zu Koln Novel Marker Genes for Regulatory T Cells from Human Blood
US20120244171A1 (en) * 2011-02-15 2012-09-27 Immunogen, Inc. Cytotoxic benzodiazepine derivatives
US20180333503A1 (en) * 2017-05-16 2018-11-22 Immunogen, Inc. Anti-folr1 immunoconjugates and anti-pd-1 antibody combinations
US20220119519A1 (en) * 2018-08-08 2022-04-21 Orionis Biosciences, Inc. Sirp1a targeted chimeric proteins and uses thereof
WO2020176718A1 (fr) * 2019-02-28 2020-09-03 Shattuck Labs, Inc. Polythérapies
US20200362029A1 (en) * 2019-04-29 2020-11-19 ImmnoGen, Inc. Biparatopic fr-alpha antibodies and immunoconjugates
WO2023086929A1 (fr) * 2021-11-12 2023-05-19 Shattuck Labs, Inc. Protéines chimériques à base de sirp1a et cd40l

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