WO2025168802A1 - Récepteur antigénique chimérique se liant à ca125 pour le traitement du cancer de l'ovaire - Google Patents

Récepteur antigénique chimérique se liant à ca125 pour le traitement du cancer de l'ovaire

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Publication number
WO2025168802A1
WO2025168802A1 PCT/EP2025/053298 EP2025053298W WO2025168802A1 WO 2025168802 A1 WO2025168802 A1 WO 2025168802A1 EP 2025053298 W EP2025053298 W EP 2025053298W WO 2025168802 A1 WO2025168802 A1 WO 2025168802A1
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cells
car
cell
muc16
seq
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Inventor
Sébastien WÄLCHLI
Else Marit INDERBERG
David Warren
Nicholas P. CASEY
Emmanuelle BENARD
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Oslo Universitetssykehus hf
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Oslo Universitetssykehus hf
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present disclosure is related to the field of cancer therapy.
  • it relates to novel targeting units and chimeric antigen receptors (CARs) comprising them, nucleic acids encoding the targeting units, nucleic acids encoding the CARs, immune cells expressing the CARs and their utility for treatment of cancer.
  • Background Ovarian cancer (OC) is the eighth most common cancer in women globally, for both incidence and deaths (1), with a 5-year survival rate of 49.7% (2, 3). This is driven by a high frequency of late-stage diagnoses - when OC is particularly aggressive - the development of chemoresistance, and evasion of host immune responses (4).
  • Standard treatment consists of cytoreductive surgery combined with platinum-based chemotherapy.
  • HGSC High-grade serous tubo-ovarian carcinoma
  • HGSC is classified into three different immunological phenotypes; desert, excluded, and inflamed ovarian tumours, based on the degree of T-cell infiltration into the epithelial compartment (7).
  • Immunotherapy in the form of immune checkpoint inhibitors performs poorly in OC (8).
  • CAR T-cell therapy faces challenges similar to adoptive cell therapy in general (9), but induction of a strong anti-tumour response, plus the inclusion of additional factors in the engineered cells (e.g. 4 th generation CARs) should help overcome these factors (10).
  • additional factors in the engineered cells e.g. 4 th generation CARs
  • debulking surgery and platinum-based chemotherapy are the treatment cornerstones, though recurrence is common.
  • new immunotherapeutic strategies are needed.
  • Chimeric Antigen Receptor (CAR) T-cell therapy empowers patients’ own T-cells to fight and eradicate cancer, and has been tested against various targets in OC.
  • a promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer-antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC.
  • CA125 cancer-antigen 125
  • the normal biological function of MUC16 is to provide a protective lubricating barrier at mucosal surfaces, but in cancer it can facilitate peritoneal metastasis by promoting proliferation and inhibiting apoptosis (13, 14).
  • MUC16 is a single-pass integral membrane glycosylated mucin protein, with a large extracellular region comprising multiple tandem repeats. Cleavage and release of this extracellular domain results in the generation of the Cancer Antigen 125 (CA125) (15).
  • CA125 Cancer Antigen 125
  • This cleaved CA125 has three main topographic domains, can be detected in the serum of OC patients, and is used as a diagnostic marker for OCs (16, 17).
  • cells expressing CARs comprising the claimed targeting units can be useful as such, but they can also complement other therapeutic approaches targeting MUC16ecto.
  • the cells provided herein could be included in an earlier line of treatment than treatments based on MUC16 ecto .
  • the present disclosure provides a method of treatment of MUC16-positive ovarian cancer or MUC16-positive pancreatic cancer comprising the steps, in any order: a) administering a cell expressing a CAR for specific binding to an epitope located on CA125 of MUC16 to a patient in need thereof, wherein the CAR comprises a scFv represented by any one of SEQ ID NO: 11 to 16, or a scFv represented by a sequence with more than 90% identity thereto (i.e. to any one of them), and b) administering a cell expressing a CAR for specific binding to an epitope located on MUC16ecto to the patient.
  • Physiological conditions means the environment encountered or simulated, in a living human patient, where the disclosed proteins are intended to bind an epitope located on human target-cells.
  • physiological conditions in most human extracellular fluids are normally ca. 37°C, pH in the range of 6.0 to 7.5.
  • the target-cells are cancer cells in a solid tumor
  • the physiological conditions are the ones found in the solid tumor, i.e. the tumor microenvironment (TME).
  • TME tumor microenvironment
  • the TME is often hypoxic and acidic.
  • MUC16 has been referred to as CA125 in the prior art.
  • CA125 refers to the part of MUC16 which tends to be cleaved off from cancer cells.
  • Chimeric antigen receptors are well known.
  • a CAR comprises, from N-terminal to C-terminal, an Fv such as a scFv, optionally a hinge, a transmembrane domain, at least one co-stimulatory domain and a signaling domain such as CD3 ⁇ . It has been speculated that soluble CA125 may interfere with CAR reactivity, by binding to the CAR in the absence of target-cells (18). These concerns are partially supported by the observations of interference caused by shedding of other CAR targets (e.g. 19).
  • VL CDR1 (SEQ ID NO: 1): QSLLYTSIQKNY VL CDR2 (SEQ ID NO: 2): WAS VL CDR3 (SEQ ID NO: 3): QQYYTYPWT VH CDR1 (SEQ ID NO: 4): GYTFTDYY VH CDR2 (SEQ ID NO: 5): INPNNGDN VH CDR3 (SEQ ID NO: 6): AKDGDYAMDY Framework sequences are structurally conserved regions that normally tend to form a ⁇ -sheet structure positioning the CDRs for specific binding to the target epitope under physiological conditions.
  • the first framework sequence is N-terminal to the CDR1
  • the second framework sequence is located between CDR1 and CDR2
  • the third framework sequence is located between CDR2 and CDR3. Accordingly, both a VL and VH can be roughly visualized as follows, with the CDRs boxed and the N-terminal indicated as N-: N-FRAMEWORK1CDR1FRAMEWORK2CDR2FRAMEWORK3CDR3FRAMEWORK4
  • the antigen binding unit comprises or consists of VL-linker-VH (from N- to C-terminus). In another embodiment, the antigen binding unit comprises or consists of VH-linker-VL (from N- to C- terminus).
  • Such antigen binding units are often referred to as single chain Fv’s (scFv’s).
  • the linker needs a certain length in order to allow the VH and VL to form a functional antigen binding unit.
  • the linker comprises 10 to 30 amino acid residues.
  • the linker comprises 15 to 25 amino acid residues, in particular glycine and/or serine residues.
  • the linker is a G4S linker, i.e. a peptide linker comprising repeating units with the sequence GGGGS.
  • suitable linkers may comprise 3, 4 or 5 adjoining repeating G4S units.
  • the framework sequences may tolerate variation without destroying the specificity and affinity to the target antigen.
  • substitutions of amino acid residues may be tolerated better than deletions or additions of amino acid residues.
  • Replacing murine framework sequences with human framework sequences, preferably of similar length is known as humanization.
  • the term "conservative amino acid substitution”, as used herein, refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acids with similar side chains tend to have similar properties, and thus a conservative substitution of an amino acid important for the structure or function of a polypeptide may be expected to affect polypeptide structure/function less than a non-conservative amino acid substitution at the same position. 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. asparagine, glutamine, serine, threonine, tyrosine), non-polar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).
  • acidic side chains e.g. aspartic acid, glutamic acid
  • uncharged polar side chains e.g. asparagine, glutamine, serine, threonine, tyrosine
  • non-polar side chains e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline,
  • each VL and VH herein comprises three CDRs flanked by human framework sequences. Human framework sequences are structurally conserved regions that normally tend to form a ⁇ -sheet structure delicately positioning the CDRs for specific binding to the target antigen under physiological conditions.
  • human framework sequences are available from known human antibodies and from the international ImMunoGeneTics information system (IMGT) online database (see Giudicelli et al, Nucleic Acids Research, 2006, Vol.34, Database issue D781-D784), but the term also covers human framework sequences comprising amino acid substitutions.
  • Each of the human framework sequences may optionally comprise 0 to 5 amino acid substitutions relative to the natural sequence.
  • An amino acid substitution is a sequence wherein an amino acid residue in a specific position is substituted for a different amino acid residue at the corresponding position, apparent when the sequences are aligned.
  • Each of the human framework sequences may optionally comprise 1 amino acid substitution.
  • Each of the human framework sequences may optionally comprise 2 or up to 2 amino acid substitutions.
  • Each of the human framework sequences may optionally comprise 3 or up to 3 amino acid substitutions.
  • Each of the human framework sequences may optionally comprise 4 or up to 4 amino acid substitutions.
  • Each of the human framework sequences may optionally comprise 5 or up to 5 amino acid substitutions.
  • the substitutions may be conservative substitutions. Even if such framework sequences are not necessarily previously known from human antibodies, they may provide lower immunogenic risk compared to a murine framework sequence. In one embodiment, 0 to 5 amino acid residues in the human framework sequences are substituted with the corresponding amino acid residue(s) from the murine parent sequences.
  • scFv’s comprising CDRs from a murine antibody and human framework sequences which each may optionally comprise 0 to 5 substitutions, are referred to as humanized scFv’s.
  • the human framework sequences are mature human framework sequences available from known human antibodies. Without being bound by theory, such framework sequences may convey very low risk of triggering unwanted immunogenic responses against the antigen binding unit, and at the same time increase the likelihood of obtaining stable binding units which are expressed well in cellular systems.
  • the CDRs are retained, as in the parental VH and VL sequences.
  • CDR identification schemes may yield different CDRs. For example, they may be shorter or longer, or positioned slightly differently in the VH or VL sequences (e.g. in a second scheme the CDR may be partially displaced up- or downstream relative to a first scheme).
  • the CDRs shall be determined using the well-known IMGT system. Humanization may involve grafting CDRs from murine VL and/or VH into selected human VL and/or VH, i.e. replacing the human CDRs.
  • human framework sequences may replace murine framework sequences in the murine VL and/or VH, and thus retain the murine CDRs.
  • humanized targeting units suitable for specific binding to an epitope located on CA125 of MUC16 comprising a VL selected from the group consisting of SEQ ID NO: 18, 20, 22, 24 and 26 or sequences with more than 80% sequence identity thereto, and a VH selected from the group consisting of SEQ ID NO: 19, 21, 23, 25 and 27 or sequences with more than 80% sequence identity thereto, provided -the VL comprises CDR1, CDR2 and CDR3 represented by SEQ ID NO: 1, 2 and 3, respectively, and -the VH comprises CDR1, CDR2 and CDR3 represented by SEQ ID NO: 4, 5 and 6, respectively.
  • humanized targeting units suitable for specific binding to an epitope located on CA125 of MUC16, comprising a VL selected from the group consisting of SEQ ID NO: 18, 20, 22, 24 and 26 or sequences with more than 90% sequence identity thereto, and a VH selected from the group consisting of SEQ ID NO: 19, 21, 23, 25 and 27 or sequences with more than 90% sequence identity thereto, provided -the VL comprises CDR1, CDR2 and CDR3 represented by SEQ ID NO: 1, 2 and 3, respectively, and -the VH comprises CDR1, CDR2 and CDR3 represented by SEQ ID NO: 4, 5 and 6, respectively.
  • scFv may be linked or conjugated to a therapeutic or diagnostic agent, or to a carrier which comprises or contains a therapeutic or diagnostic agent.
  • a therapeutic agent is an agent used in therapy. By therapy is meant the treatment or prevention of a disease.
  • the therapeutic agent may be an agent useful in the treatment of a neoplastic condition, particularly cancer.
  • recombinant nucleic acid molecules encoding the proteins.
  • the nucleic acid molecules provided herein may be an isolated nucleic acid molecule and may include DNA or RNA or chemical derivatives of DNA or RNA.
  • the term "nucleic acid molecule" specifically includes single- and double-stranded forms of DNA and RNA.
  • the nucleic acid (e.g. DNA or RNA) may be circular or linear.
  • a “recombinant” nucleic acid molecule is a nucleic acid molecule synthesized using recombinant techniques, e.g. molecular cloning.
  • the recombinant nucleic acid molecules provided herein may encode the protein provided herein.
  • such recombinant nucleic acids may have promoters allowing expression of the proteins in cellular systems.
  • the recombinant nucleic acid molecules or constructs provided herein may be provided within a vector.
  • vector refers to a vehicle into which the nucleic acid molecule or construct provided herein may be introduced (e.g.
  • the term “linked” as used herein with respect to the construct may simply mean that the nucleic acid molecule is directly joined to a heterologous nucleic acid sequence.
  • the nucleic acid molecule provided herein is operatively linked to a heterologous expression control sequence.
  • expression control sequence refers to nucleotide sequences located upstream of, within, or downstream of a coding sequence, and which influence transcription, RNA processing or stability, or translation of the associated coding sequence (i.e. which influence any aspect of expression of the encoded specific binding molecule). Expression control sequences include promoters, operators, enhancers, and other such cis-elements.
  • the recombinant nucleic acid molecule comprises a cDNA molecule.
  • cDNA as used herein is meant cDNA in its true and original sense (i.e. DNA synthesized by reverse transcription of mRNA), DNA amplified from original cDNA, and also DNA that is equivalent to cDNA.
  • DNA that is equivalent to cDNA is DNA that encodes a protein as provided herein and that lacks introns, such that it resembles a protein-coding sequence obtained by reverse transcription of mRNA.
  • the nucleic acid molecule encoding a protein as provided herein is codon-optimized, in particular the binding protein may be encoded by a codon-optimized cDNA sequence.
  • a or “an” entity refers to one or more of that entity; for example, “a cell”, is understood to represent one or more cells, and the term of course includes cell populations. As any skilled person will understand, a cell expressing a CAR will normally express numerous such CARs. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
  • an amino acid sequence having at least x% identity to a second amino acid sequence means that x% represents the number of amino acid residues in the first sequence which are identical to their matched amino acid residues of the second sequence when both sequences are optimally aligned via a global alignment, relative to the total length of the second amino acid sequence. Both sequences are optimally aligned when x is maximum by using the comparison matrix BLOSUM62 with gap costs: existence 11, extension 1.
  • the pharmaceutical compositions herein can be a composition suitable for administration of therapeutic proteins, nucleic acids or cells to a patient. Accordingly, said pharmaceutical compositions may for example be sterile aqueous solutions with a neutral pH.
  • said pharmaceutical compositions may for example be sterile aqueous solutions with a physiological pH.
  • Suitable cells for expressing the CARs herein include T-cells and NK-cells, but other immune cells can also be used. It is generally preferred to use immune providing at least one effector function (e.g. cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). Such cells thus include T- lymphocytes, in particular cytotoxic T-cells and helper T-cells. Other immune cells include NK cells, NKT cells, neutrophils, and macrophages. The cells may be primary cells or cell lines.
  • the pharmaceutical compositions herein can be a composition suitable for administration of therapeutic cells to a patient.
  • the most common administration route for therapeutic cells is intravenous administration.
  • said pharmaceutical compositions may for example be sterile aqueous solutions with a neutral pH.
  • said pharmaceutical compositions may for example be sterile aqueous solutions with a physiological pH.
  • the sterile pharmaceutical compositions may be a cell suspension for infusion comprising from 1 x 106 cells to 1 x 10 10 cells, such as 1 x 10 7 cells to 1 x 10 9 cells.
  • Such compositions can be for example be supplied in an infusion bag containing approximately 30 to 100 mL of a frozen suspension of the cells in 5% DMSO and 2.5% human serum albumin.
  • the most common administration route for T-cells is intravenous administration.
  • peripheral blood mononuclear cells may be obtained via a standard leukapheresis procedure.
  • the mononuclear cells may be enriched for T- cells, before transducing them with a lentiviral vector or mRNA encoding the proteins disclosed herein, in particular CARs. Said cells may then be activated with anti-CD3/CD28 antibody coated beads.
  • the transduced T-cells may be expanded in cell culture, washed, and formulated into a sterile suspension, which can be cryopreserved. If so, the product is thawed prior to administration.
  • Antibodies from three hybridomas from our hospital’s collection (33) were adapted to a CAR format.
  • K101CAR induced the strongest response against MUC16-positive (MUC16pos) tumour cells in vitro and was selected for further testing.
  • Soluble CA125 did not interfere with K101CAR T-cell cytotoxicity, and the functional activity of this CAR was superior to existing anti-MUC16 ecto CAR in vitro, and showed significant impairment of tumour growth in vivo.
  • K101CAR represents a promising candidate for clinical CAR-based therapy.
  • OC is a common gynaecological cancer with high mortality rates. This is due in part to typically late diagnosis, limited treatment options, and high rates of recurrence (4).
  • Conventional therapies are limited in efficacy and range, and new treatment modalities are needed.
  • CAR therapy has the potential to direct immune cells to targets in immunologically-cold tumour microenvironments.
  • a CAR that targets CA125 – the cleaved domain of MUC16 - can efficiently recognize and kill MUC16 pos tumours. This also confirms the re-association of CA125 with tumour cells at levels sufficient to trigger a cytotoxic response (31).
  • Another argument precluding the targeting of CA125 was the presence of the soluble antigen in the patient serum which could inhibit a CAR by competing with the cellular CA125. While the replication of “true” in situ conditions can be challenging, we also show that high levels of soluble CA125 in the immediate vicinity of the target-cells did not affect CAR T-cell activity in vitro.
  • OVCAR3 cells which are often used as a validation model, do present all the expected features; they are slow growing and MUC16 high , and, as expected, were easily killed by the anti-MUC16CARs.
  • the cervical tumour-derived HeLa cells were markedly harder to control, especially in 2D culture. We suspect that this was due to [1] heterogeneity of MUC16 expression for which we confirmed the reports of previous authors (54), and/or [2] the influence of culture conditions on CA125 expression and distribution (55).
  • K101CAR T-cells could kill HeLa cells grown in 3D and in vivo, these observations challenge the validity of the in vitro systems widely used by the CAR community, while supporting previous propositions that 3D systems are more representative or predictive of the in vivo context (56, 57).
  • K101CARs typically showed stronger reactivity than did the 4H11CARs in vitro, although the nature of the samples prevented us from directly comparing cytotoxicity against them.
  • testing of the K101CAR in vivo in an orthotopic PDX model confirmed killing under these conditions.
  • CA125 is detectable on tumour cells lines and primary tumour samples, at levels sufficient for targeting by CAR T-cells both in vitro and in vivo.
  • soluble CA125 even at high concentrations, had any effect on CAR T-cell cytotoxicity nor specificity in vitro.
  • the efficient killing of MUC16 pos target-cells by the K101CAR T-cells indicates that the extracellular repeat domain is indeed an attractive target.
  • cytotoxic immune cells expressing a CAR for specific binding to an epitope located on CA125 of MUC16, as provided herein, in their cell membrane, and a CAR targeting MUC16ecto in their cell membrane will represent a robust cell- therapeutic approach for ovarian cancer. Because cells expressing the CARs comprising the targeting unit herein were not significantly hampered by the presence of soluble CA125, such cells can provide cytotoxic effect to the cancer cells expressing MUC16 even if CA125 is cleaved off.
  • the present disclosure thus provides alternative solutions for treatment of OC based on targeting of MUC16 even in case of partial loss of CA125 antigen.
  • the present disclosure provides a method of treatment of ovarian cancer comprising the steps, a) administering a therapeutic protein, or a nucleic acid encoding it, for specific binding to an epitope located on CA125 of MUC16 to a patient in need thereof and subsequently b) administering a therapeutic protein, or a nucleic acid encoding it, for specific binding to an epitope located on MUC16 ecto to the patient.
  • the present disclosure provides a method of treatment of ovarian cancer comprising the steps, a) administering a cell expressing a CAR for specific binding to an epitope located on CA125 of MUC16 to a patient in need thereof, wherein the CAR comprises a scFv as provided herein, and subsequently b) administering a cell expressing a CAR for specific binding to an epitope located on MUC16ecto to the patient.
  • MATERIALS AND METHODS Analysis of RNA-Seq data RNA-Seq data from The Cancer Genome Atlas (TCGA, released July 27, 2022) project for OC for 378 tumour samples was used.
  • MUC16 Since the MUC16 gene is known to be highly expressed in OC tissues, we focussed upon a selection of genes (MUC16, MUC1, MSLN, FOLR1) in order to show the significant differential expression of MUC16 in the TCGA database (35). To visualise the results of differential expression analyses of selected genes (MUC16, MUC1, MSLN, FOLR1), pheatmap was used. Cell culture and cell lines Standard in vitro culture was at 37oC, with 5% CO2, in a humidified environment.
  • target-cells Prior to co-culture experiments, target-cells were detached with EDTA alone (5 mM in PBS, without Trypsin). Spheroids composed of tumour cells were generated using standard methods. Briefly, flat bottom 96-well plates were coated with 1% agarose dissolved in PBS. After 2 hours at room temperature, 2000 tumour cells in 200 ⁇ L of culture medium were added per well. PBMCs were isolated from whole blood of healthy donors, under an approved institutional protocol, and were cultured in X-vivo 15 (Lonza, Switzerland), supplemented with 5% human serum (TCS Biosciences, Buckingham MK182LR, UK), and IL-2 (Clinigen, UK) at 100 U/mL.
  • 24-well culture plates were pre-coated with 500 ⁇ L/well of PBS containing 0.5 ⁇ g anti-CD3 (functional grade OKT3, eBioscience, USA, #16-0037-85) and 0.5 ⁇ g anti-CD28 (functional grade CD28.6, eBioscience, USA, #16-0288-85) antibodies, for 2 hours at room temperature. This was removed, and PBMCs were added at 10 6 cells per mL, per well. After 2-3 days, cells were counted, and available for transduction. Production of retroviral vectors, transduction, and protein production Production of retroviral particles, and transduction of T-cells, were performed as described previously (36).
  • CA125 contains three topographically distinct antigenic determinants, with most monoclonal antibodies grouped as OC125-like (group A), or M11-like (group B), while OV197 alone constitutes a third category, group C (37).
  • the hybridomas producing the anti-CA125 antibodies were generated at The Department of Medical Biochemistry, Oslo University Hospital, Radium Hospital, Oslo, Norway. We selected hybridomas K93 and K95 (both group A), and K97 and K101 (both group B) (38).
  • luciferase-expressing tumour cells were co-cultured with CAR T-cells in a 96-well plate in the presence of D-Luciferin (75 ⁇ g/mL, Perkin Elmer, Norway) at indicated effector: target (E: T) ratios.
  • D-Luciferin 75 ⁇ g/mL, Perkin Elmer, Norway
  • E T
  • Cells were placed in an incubator at 37 °C, 5% CO2 and bioluminescence was measured with a luminometer (VICTOR Multilabel Plate Reader, Perkin Elmer) at several time points up to 24 hours.
  • VIP luminometer
  • target-cell lines at 2000 cells/well, were seeded as spheroids for 5 days.
  • T-cells were added (10,000 cells/well), along with Annexin V red, at the recommended concentration (Sartorius, USA, catalogue number 4641). Annexin V signal was recorded hourly, and the Total Red Object Integrated Intensity (RCU x ⁇ m2/Image) was calculated.
  • Samples were resuspended in 10% RPMI, supplemented with collagenase II (Sigma Aldrich, C6885-500MG, at 4.7 mg/mL final) and DNase (Sigma Aldrich, DN25-100MG, at 0.57 mg/mL final), and incubated at 37 o C with constant rotation for 1 hour, or longer if necessary. Samples were then passed successively through 100 ⁇ m, 70 ⁇ m, and 40 ⁇ m cell strainers. Samples were centrifuged at 500xg (5 minutes), then resuspended in 5 mL ACK lysis buffer (Lonza, #10-548E) for 30 seconds, before 5 mL of 10% RPMI was added.
  • collagenase II Sigma Aldrich, C6885-500MG, at 4.7 mg/mL final
  • DNase Sigma Aldrich, DN25-100MG, at 0.57 mg/mL final
  • Samples were centrifuged at 400xg (10 minutes), then resuspended and frozen, or used fresh.
  • the samples were labelled with commercial anti-CA125 antibody (X75 at 1/200 dilution. Thermo Fisher, #MA1-90039) followed by a secondary antibody (APC goat anti-mouse IgG, BioLegend #405308, at 1/200 dilution), and labelling intensity was normalised against the same samples labelled with the secondary antibody only.
  • samples were grouped as CA125-low/negative, medium, or high.
  • CAR T-cells and patient samples were co-cultured overnight at an E: T ratio of 1:2, in the presence of anti-CD107a (BD Bioscience, USA, catalogue number 555802).
  • CD107a labelling was compared to CAR T-cells cultured under the same conditions, but without patient-cells present.
  • PET-CT imaging and analysis were acquired using the integrated nanoScan PC PET/CT (Mediso Ltd, Hungary) featuring spatial resolution of 800 ⁇ m and 300 ⁇ m of the PET and CT detector systems, respectively.
  • the field of view (FOV) of the stacked images was 9.6 axial ⁇ 10 cm transaxial allowing whole-body 3D imaging of the mice. Animals were scanned using a dual mouse bed with integrated heating (37 °C).
  • Each PET scan was conducted over 20 minutes, 45 minutes after intravenous administration of 18 F-Fluorodeoxyglucose ( 18 F-FDG; 5-12 MBq/mouse).
  • 18 F-FDG 18 F-FDG
  • a whole-body CT scan helical projections with tube energy of 50 kVp, exposure time 300 ms, 7.2 projections, max FOV, binning 1-4
  • PET images were reconstructed using the Nucline software and parameters were static normal, whilst CT images were reconstructed using a RamLak filter. The PET and CT images were co-registered automatically.
  • a signal above 2.5 SUV was regarded as a true tumour signal and a spherical volume of interest (VOI) was drawn semi-automatically for estimation and calculation of SUV mean and SUVmax.
  • Soluble CA125 To represent cleaved CA125 in our experiments, we used human calibration-grade CA125 protein (#abx060961, Abbexa, UK). Where this was used in co-culture assays (BLI cytotoxicity and CD107a functional assays), it was added in solution, at the indicated concentrations. Dot Blots Proteins of interest were applied to a nitrocellulose membrane and allowed to dry.
  • the membrane was then blocked with 5% Bovine Serum Albumin (BSA) in Tris- Buffered Saline (TBS), before incubation with the primary antibody (various).
  • BSA Bovine Serum Albumin
  • TBS Tris- Buffered Saline
  • the membrane was washed three times in TBS-Tween, then incubated with Horse Radish Peroxidase-conjugated anti-mouse IgG secondary antibody (#62-6520, ThermoFisher). After another three washes with TBS-Tween, the membrane was incubated for 1 minute with Enhanced ChemiLuminescent substrate, then the luminescent signal was read on an Amersham Imager 600 (G.E. Healthcare, USA). Semi-quantitative analysis was performed using GelQuant software (BiochemLabSolutions.com).
  • mice Female NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ, OUS and UiB) and NXG (NOD- Prkdc scid -IL2rg Tm1 /Rj, OUS) mice were maintained in pathogen-free conditions under the respective institutional animal care protocols. All animal experiments were conducted in compliance with the procedures from the Norwegian State Commission for Laboratory Animals and approved by the Norwegian Food Safety Authority. For the cell-line study, luciferase-expressing OVCAR3 or HeLa cells were trypsinised and washed twice in PBS.
  • mice Six- to 10-week-old NXG mice were injected intraperitoneally (i.p.) with 1x10 6 luciferase-expressing OVCAR3 or HeLa cells in 200 ⁇ l PBS. After 3 days, 200 ⁇ L D-luciferin (20 mg/mL, #122799-5, Perkin Elmer, Waltham, USA) was injected i.p., and engraftment was confirmed by bioluminescence using the IVIS Spectrum In Vivo Imaging System (Perkin Elmer). Mice were allocated to treatment groups based on comparable tumour loads. The same day 5x10 6 T-cells (Mock, K101CAR, or 4H11CAR) were injected i.p.
  • MUC16 is expressed in a large proportion of OC samples Although already recognized as a valid OC target, MUC16 distribution was analysed from a TCGA cohort of 375 ovarian tumour samples (46). We compared MUC16 expression to that of other OC-validated targets (MSLN, FOLR1 and MUC1) (47-49) and included the clinical stage information. We conducted DESeq2 analysis on the TCGA tumor samples, confirming that MUC16 was expressed in the majority of OC samples.
  • MUC16 was compared to the three other targets but none of these genes were in the top 100 for RNA expression, they however all show positive fold changes throughout the different clinical staging groups. These data confirm that MUC16 is a marker for OC and therefore a potential drug target for treating advanced patients.
  • Anti-CA125 scFv leads to functional CAR constructs Supernatants from four anti-CA125 hybridomas (K-series, (33)) were tested against CA125-positive and CA125-negative cell lines. The commercial anti-CA125 antibody, X75, was included for reference. Labelling with each of the supernatants of the K series closely matched the commercial X75 antibody signal.
  • K95 and K101CARs demonstrated a superior activity toward OVCAR3 and HeLa cells and no reactivity against the MUC16-negative (MUC16 neg ) HEK cells.
  • K95CAR T-cells reacted against MUC16 neg BL41 lymphoma cell line and was therefore eliminated from the study.
  • K93CAR showed weak reactivity in the different assays and further development was abandoned.
  • K101CAR T-cells In order to validate the robustness of K101CAR T-cells, we tested them against a series of primary patient samples, including peritoneal effusions and samples derived from debulking surgery of solid tumours. MUC16 expression was measured by anti-CA125 (X75) labelling and correlated to MUC16CAR T-cell reactivity assessed by degranulation and anti-CD107a staining. From a total of 6 patient-derived samples, we observed that K101CAR T-cells reactivity followed MUC16 presence, whereas 4H11CAR T- cells did not react with these samples. From these data K101CAR appears more potent than 4H11CAR to redirect T-cells against MUC16 pos targets.
  • Soluble CA125 does not alter K101CAR T-cell activity
  • the commercial X75 antibody was used as a positive control and the background level was fixed using an irrelevant hybridoma supernatant, producing an anti-CEA antibody, CEA10.
  • CEA10 anti-CEA antibody
  • K101 also reacts with CA125 in a cell-free context, suggesting a risk of potential inhibition of K101CAR by serum CA125.
  • K101CAR T-cells are functional even at a high concentration of CA125 and therefore not sensitive to the presence of soluble target.
  • K101CAR T-cells are efficient in vivo
  • T-cells were activated, transduced, then expanded in vitro, with cell proliferation and CAR expression monitored to ensure that CAR T-cell populations were comparable.
  • mice After intraperitoneal (i.p.) engraftment of the slow growing OC cell line OVCAR3, immunodeficient mice received two injections (days 3 and 11) of CAR T-cells or Mock T-cell controls.
  • OVCAR3 form numerous small, solid tumours, with considerable expansion of ascites in the peritoneal space.
  • the mice receiving Mock T-cells developed a high tumour burden in approximately 30 days, whereas very little signal was detected in mice receiving the MUC16CAR T-cells until around day 95. Accordingly, we observed prolonged survival compared with the Mock T-cell group.
  • 4/5 of the mice treated with MUC16CAR T-cells were alive at the 4-month endpoint of the experiment.
  • K101CAR T-cells were performing in a complex model.
  • PDX26 patient-derived cells
  • Kandalaft LE Dangaj Laniti D, Coukos G. Immunobiology of high-grade serous ovarian cancer: lessons for clinical translation. Nature Reviews Cancer.2022;22(11):640-56. 11. Benard E, Casey NP, Inderberg EM, Walchli S. SJI 2020 special issue: A catalogue of Ovarian Cancer targets for CAR therapy. Scand J Immunol.2020;92(4):e12917. 12. Bast RC, Jr., Klug TL, St John E, Jenison E, Niloff JM, Lazarus H, et al. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl J Med.

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Abstract

La présente divulgation concerne des unités de ciblage pour une liaison spécifique à CA125. Ces unités de ciblage sont appropriées pour une mise en oeuvre dans des récepteurs antigéniques chimériques (CAR). Contrairement à ce qui a été prédit précédemment, il a été découvert que les CAR comprenant de telles unités de ciblage peuvent reconnaître et tuer efficacement les tumeurs MUC16pos.
PCT/EP2025/053298 2024-02-09 2025-02-07 Récepteur antigénique chimérique se liant à ca125 pour le traitement du cancer de l'ovaire Pending WO2025168802A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
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WO2011119979A2 (fr) 2010-03-26 2011-09-29 Memorial Sloan-Kettering Cancer Center Anticorps anti-muc16 et leurs procédés d'utilisation

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WO2011119979A2 (fr) 2010-03-26 2011-09-29 Memorial Sloan-Kettering Cancer Center Anticorps anti-muc16 et leurs procédés d'utilisation

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