EP4022313A1 - Évaluation immunohistochimique de l'expression de b7-h3 - Google Patents

Évaluation immunohistochimique de l'expression de b7-h3

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Publication number
EP4022313A1
EP4022313A1 EP20765196.9A EP20765196A EP4022313A1 EP 4022313 A1 EP4022313 A1 EP 4022313A1 EP 20765196 A EP20765196 A EP 20765196A EP 4022313 A1 EP4022313 A1 EP 4022313A1
Authority
EP
European Patent Office
Prior art keywords
antibody
minutes
antigen
binding fragment
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20765196.9A
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German (de)
English (en)
Inventor
Sonia SEQUEIRA
Ahmed Mahiuddin
Claus J. MØLLER SAN-PEDRO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Y Mabs Therapeutics Inc
Original Assignee
Y Mabs Therapeutics Inc
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Filing date
Publication date
Application filed by Y Mabs Therapeutics Inc filed Critical Y Mabs Therapeutics Inc
Publication of EP4022313A1 publication Critical patent/EP4022313A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a method for providing or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof using immunohistochemical methods.
  • the present invention further relates to an immunohistochemical-method for detecting and optionally quantifying overexpression of B7-H3 for the selection of patients that may benefit from B7-H3 antibody or antigen binding fragment thereof based radioimmunotherapy, immunotherapy or cell therapy. More specifically, the invention relates to a method for providing or measuring a biomarker for assessing the efficacy and/or safety of an antibody or antigen-binding fragment thereof based on B7-H3 expression in the treatment of cancer in an individual, or in the treatment of a specific indication of cancer, such as ovarian cancer.
  • the invention further discloses a method for measuring or qualifying the clinical benefit of a B7-H3 antibody or antigen- binding fragment thereof. Additionally, the invention discloses a method for detecting B7-H3 expression in a tissue sample comprising cancer cells by use of a murine 8H9 antibody. In addition, the invention relates to a kit of parts adapted to carry out the inventive method. Moreover, the invention relates to a method of treating a cancer, comprising providing or measuring a biomarker for measuring and/or verifying the suitability, efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody is used in said treatment.
  • a particular utility comes from demonstrated clinical benefit with radio immunotherapies for high expressing B7H3 tumors and the need to determine benefit and select patients with heterogeneous expression (among patients and among tumor tissues, as well as localization in tumor tissue).
  • Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3 positive tumors.
  • Murine 8H9 antibody targeting B7-H3 protein is broadly reactive with human solid tumors, including embryonal tumors and carcinomas. It has shown favorable tumor uptake for both sarcomas and brain tumors in xenograft models. When conjugated to cobra venom factor, it induces efficient complement-mediated tumor lysis. In its single chain Fv (scFv) format, it targets a potent immunotoxin to sarcoma and glioma in preclinical models. As a chimeric antigen receptor, it redirects natural killer cells to kill B7-H3 positive tumor cells.
  • scFv single chain Fv
  • radiolabeled murine 8H9 antibody was shown to prolong survival among high risk patients with solid tumors suffering from central nervous system (CNS) metastasis. It is a promising target for radioimmunotherapy of leptomeningeal metastases, diffuse intrinsic pontine glioma and peritoneal metastases (Ahmed et al. Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3, The Journal of Biological Chemistry Vol. 290, NO. 50, pp. 30018 - 30029, December 11, 2015).
  • Murine mAb 8H9 has been shown to bind to a unique epitope on both the 2lg- and 4lg-B7-H3 isoforms of B7-H3, namely the FG-loop that includes the "I RDF " sequence motif (Ahmed et al. Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3, The Journal of Biological Chemistry Vol. 290, NO.
  • B7-H3 is a new cancer-specific endothelial marker in clear cell renal cell carcinoma, OncoTargets and Therapy 2013:6 1667-1673, 2013
  • RCC clear cell renal cell carcinoma
  • B7-H3 antibody goat anti-human B7-H3 monoclonal antibody from R&D Systems, Minneapolis, MN, USA
  • Expression patterns of B7-H3 were compared with that of the pan-endothelial cell specific marker CD34. Paired normal renal parenchyma specimens from all patients and four corpus luteum specimens were also analyzed.
  • Formalin-fixed paraffin-embedded tissues were cut into 5 mm sections, deparaffinized and rehydrated in a graded series of ethanols.
  • Antigen retrieval was done heating tissue sections in ethylenediaminetetraacetic acid (EDTA) 1 mmol/L, pH 8 to 121 °C using a Digital Decloing Chamber, cooling to 90 °C, and incubating for 5 minutes, and treating with peroxidase blocking reagent. Sections were incubated with a biotin-labeled polyclonal antibody against B7-H3 and CD34 followed by a horseradish peroxidase-conjugated anti- biotin antibody and visualization by diaminobenzidine staining.
  • EDTA ethylenediaminetetraacetic acid
  • Sections were lightly counterstained with hematoxylin.
  • the study detected B7-H3 expression in cancer cells in 19% of clear cell RCC cases, and B7-H3 expression in tumor vasculature was confirmed in 98% of cases.
  • the study states that the total probability of cancer cell-specific B7-H3 expression in the clear cell RCC population was ⁇ 20%, suggesting that induction of cytotoxicity or antitumor immunity by agents against B7-H3 expressed in cancers cells may not be sufficient in clinical practice.
  • B7-H3 is related to tumor progression in ovarian cancer
  • Oncology reports 38, 2017 shows that B7-H3 affect ovarian cancer progression through the Jak2/Stat3 pathway, indicating that B7-H3 has the potential to be a useful prognostic marker.
  • Paraffin embedded samples from biopsies have been used in immunohistochemical (IHC) methods in combination with goat anti-human B7-H3 antibody as described in Qin et al, and with mouse anti-human B7-H3 antibody as described in Loos et al. Further, frozen samples have been used in immunohistochemical methods. However, paraffin embedded samples in combination with a murine 8H9 antibody, such as an antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2, have not been used successfully in this context. Because frozen tissue samples have limited storage stability, and because paraffin embedded tissue samples in general preserve tissue better, there is a need for a method that works on paraffin embedded tissue samples. The present invention discloses a method that can use both frozen samples and paraffin embedded samples, and which may find general use within the field of IHC.
  • An IHC technique may be used for each individual patient. Multiple samples may be collected from an individual patient and analyzed for the expression of B7-H3.
  • B7-H3 is highly expressed.
  • B7-H3 is not necessarily highly expressed in other types of cancer.
  • Zang et al. has shown that ovarian borderline tumors and carcinomas aberrantly express B7-H3.
  • the test of the invention may help to determine the highest possible dose for a population or individual who may benefit from treatment targeting B7-H3.
  • an immunohistochemical method which allows measurement, detection and/or evaluation of a biomarker for the efficacy and/or the safety and/or clinical benefit of specific antibodies, such as B7-H3 antibodies.
  • a method to detect which patient might benefit from B7-H3 antibody or antigen binding fragment thereof based radioimmunotherapy, immunotherapy or cell therapy there is a need for such a method, which may be used on at least one paraffin embedded tissue sample or a sample formerly embedded in paraffin.
  • the anti-B7-H3 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof disclosed in International Publication No. WO 02/32375, WO 2003/033670, WO 03/075846, WO 2008/116219, WO 2016/033225 and WO 2018/209346, all of which are incorporated by reference in their entireties.
  • the invention concerns a method for detecting expression and/or overexpression of an antigen in at least one tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample is selected among a thawed sample, a frozen sample, a fresh sample, a paraffin embedded sample, a formalin embedded sample, and/or a sample which has formerly been embedded in formalin and/or paraffin.
  • a “thawed sample” is a sample which has been frozen, but is no longer frozen.
  • a “paraffin embedded sample” is a sample that is embedded in paraffin.
  • a “formalin embedded sample” is a sample that is embedded in formalin.
  • a sample may belong to more than one category, e.g. a paraffin embedded sample may also be a thawed sample, and a sample that is embedded in formalin may also be embedded in paraffin.
  • the invention concerns a method for detecting expression and/or overexpression of an epitope in a tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample preferably is a sample which has formerly been embedded in formalin and/or paraffin, and wherein said method comprises the steps of: i. Enzyme blocking with an enzyme blocking substance, ii. Protein blocking with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
  • the invention concerns the method, wherein said epitope is an epitope of B7-H3 antigen. It has been shown by indirect evidence that 8H9 binds to 'IRDF' sequence in B7H3. 4lg-B7H3 (more common isoform) has 2 IRDF motifs (since 4lg B7H3 is a duplicate of two 2lg-B7H3 sequences). The 'IRDF' sequence is located on the FG-loop, a region thought to be critical to the function of B7-family of proteins.
  • the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
  • the invention concerns a method for detecting the clinical benefit of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
  • the individual is a specific individual or a specific cancer patient, as the method is to be used for each individual patient.
  • the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer, wherein the method is performed on tissue samples comprising cancer cells.
  • the invention concerns a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof, wherein said chimeric antigen receptor is used according to a method of the invention.
  • CAR chimeric antigen receptor
  • the invention concerns a CAR-T cell expressing the CAR.
  • the invention concerns a population of the CAR-T cells.
  • the invention concerns a composition comprising the population of CAR-T cells.
  • the invention concerns a method of treating cancer in an individual, wherein the method comprises providing of a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
  • the invention concerns a method for treating cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the indication, such as ovarian cancer, for treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
  • the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
  • the invention concerns a method for treating cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
  • the invention concerns a method of diagnosing cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
  • the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the diagnosis of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
  • the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said diagnosis.
  • the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method according to the invention, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
  • the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof.
  • the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
  • the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises evaluation of the clinical benefit of the antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient.
  • the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention.
  • the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof.
  • the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, and wherein the diagnosis of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method according to the invention.
  • the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises evaluation of the clinical benefit of the antibody or antigen-binding fragment thereof, comprising use of a method according to the invention, and wherein the clinical benefit is the clinical benefit for a patient.
  • the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention.
  • the invention concerns a kit of parts, adapted to carry out a method of the invention.
  • the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is to be used in the treatment of a cancer.
  • the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify the clinical benefit a patient has or will have from treatment with an antigen or antigen-binding fragment thereof, and wherein said patient is a cancer patient.
  • the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer.
  • the invention concerns a kit of parts, wherein said kit is used in detecting B7-H3 expression in a tissue sample comprising cancer cells, and wherein said sample is selected among a sample that have been embedded in paraffin and a sample have been frozen.
  • the invention concerns a method for treating cancer in an individual, wherein the method comprises use of a kit of the invention.
  • the invention concerns a method of treating ovarian cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to the invention.
  • the invention concerns a method of treating gastric cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to the invention.
  • the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
  • the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
  • the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No.
  • a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer. According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer.
  • the invention concerns a method for detecting expression and/or overexpression of an antigen in at least one tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample is selected among a thawed sample, a frozen sample, a fresh sample, a paraffin embedded sample, a formalin embedded sample, and/or a sample which has formerly been embedded in formalin and/or paraffin.
  • a "thawed sample” is a sample which has been frozen, but is no longer frozen.
  • a "paraffin embedded sample” is a sample that is embedded in paraffin.
  • a “formalin embedded sample” is a sample that is embedded in formalin.
  • a sample may belong to more than one category, e.g.
  • a paraffin embedded sample may also be a thawed sample, and a sample that is embedded in formalin may also be embedded in paraffin.
  • the invention concerns a method for detecting expression and/or overexpression of an epitope in a tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample preferably is a sample which has formerly been embedded in formalin and/or paraffin, and wherein said method comprises the steps of: i. Enzyme blocking with an enzyme blocking substance, ii. Protein blocking with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
  • the invention concerns the method, wherein said epitope is an epitope of B7-H3 antigen.
  • the invention concerns the method, wherein said primary antibody has a concentration of about 30 mg/ml, and wherein said binding with a primary antibody is carried out at about 4 °C, and/or wherein said binding is carried out for about 4- 24 hours.
  • the invention concerns the method , wherein said method further comprises the following step: i. Antigen retrieval with Tris-EDTA, wherein said Tris-EDTA has a pH value of about 9 and/or a temperature of about 100 °C
  • the invention concerns the method, wherein said antigen retrieval is carried out for about 1-30 minutes, 5-25 minutes, 10-20 minutes or preferably about 15-20 minutes.
  • the invention concerns the method, wherein said antigen retrieval is carried out for about 15 minutes.
  • the invention concerns the method, wherein said antigen retrieval is carried out for about 20 minutes.
  • the invention concerns the method, wherein said method further comprises a step of cooling down said tissue sample at room temperature, wherein said cooling down is performed after said antigen retrieval.
  • the invention concerns the method, wherein said cooling down is performed for about 1-30 minutes, about 10-25 minutes, about 15-20 minutes or preferably about 20 minutes.
  • the invention concerns the method, wherein said tissue sample is from an individual, and wherein said method is used for evaluating whether said individual may benefit from a therapy, wherein said therapy is selected among radioimmunotherapy, immunotherapy or cell therapy.
  • the invention concerns the method, wherein said antigen is a B7-H3 antigen.
  • B7-H3 is also referred to as CD276.
  • the invention concerns the method, wherein said B7-H3 antigen comprises the sequence of SEQ ID No. 17 and/or 18.
  • the invention concerns the method, wherein said B7-H3 antigen comprises at least one peptide selected among the peptide according to SEQ ID No. 15 and the peptide according to SEQ ID No. 16.
  • the invention concerns the method, wherein said method uses antibodies to detect proteins in cells of said tissue sample.
  • the invention concerns the method, wherein said primary antibody is an anti-B7H3 antibody.
  • the invention concerns the method, wherein an anti-B7-H3 antibody or antigen-binding fragment thereof is used to detect B7-H3 expression.
  • the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
  • the CDRs refers to the complementarity determining region amino acid sequences of an antibody.
  • the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
  • the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No.
  • a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
  • the invention concerns the method, wherein said antigen- binding fragment thereof is a single chain variable fragment (scFv).
  • scFv single chain variable fragment
  • the invention concerns the method, wherein said scFv comprises a portion of the amino acid sequence set forth in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
  • the 8H9 scFv gene sequence, sense strand is represented in SEQ ID No. 10, the complementary strand is represented in SEQ ID No. 11 and the cDNA is represented in SEQ ID No. 12.
  • the invention concerns the method, wherein said scFv comprises at least one of the amino acid substitutions K13E, R18Q, R45Q, K103E and K107E.
  • the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof is a murine 8H9 antibody or an antigen- binding fragment thereof.
  • the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is humanized.
  • the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
  • the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is radiolabeled.
  • the invention concerns the method, wherein said radiolabel is selected among an alpha or beta emitting radiotherapeutic label, a PET label and a SPECT label.
  • the invention concerns the method, wherein said alpha or beta emitting label is selected among 131 l, 177 Lu, 228 Th, 225 Ac, 227 Ra.
  • the invention concerns the method, wherein said PET label is selected among 124 l, 18 F, 64 Cu, 68 Ga, 11 C 225 Ac and 89 Zr.
  • the invention concerns the method, wherein said SPECT label is selected among 131 l, 177 Lu, 99 mTc, 64 Cu and 99 mTc. According to an embodiment, the invention concerns the method, wherein said antibody or antigen-binding fragment thereof this conjugated to a chelator compound.
  • the invention concerns the method, wherein said chelator compound is bound to a radioactive isotope.
  • the invention concerns the method, wherein said radioactive isotope is selected among 124 l, 131 l and 177 Lu or 99 mTc, 64 Cu (chelated to NOTA) and 89 Zr (chelated to DFO).
  • the invention concerns the method, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO.
  • the invention concerns the method, wherein said DOTA is a variant of DOTA.
  • the invention concerns the method, wherein said DTPA is a variant of DTPA.
  • DOTA is also referred to as l,4,7,10-tetraazacyclododecane-l,4,7 10-tetraacetic acid, and has the formula (CH2CH2NCH2C02H)4 DTPA is also referred to with the lUPAC name 2-[bis[2-
  • DTPA has the molecular formula C14H23N3O10.
  • the invention concerns the method, wherein said method may be used on at least a sample that has formerly been embedded in paraffin, as well as a sample which has formerly been frozen.
  • the invention concerns the method, wherein said method further comprises one or more steps of rinse, preferably wherein at least one rinsing step is with water.
  • the invention concerns the method, wherein said water is d2H20. d2H20 is also referred to as doubles-distilled water.
  • the invention concerns the method, wherein one or more or all of said steps are separated by wash with at least one wash buffer.
  • the invention concerns the method, wherein one or more of all said steps are separated by at least one wash with at least one wash buffer.
  • a wash may be defined as adding an adequate volume of a soluble reagent, preferably a buffer, to a tissue sample and removing the soluble reagent, preferably buffer, again.
  • the invention concerns the method, wherein one or more of all said steps are separated by at least two washes with at least one wash buffer.
  • the invention concerns the method, wherein one or more of all said steps are separated by at least three washes with at least one wash buffer. According to an embodiment, the invention concerns the method, wherein each wash is carried out for about 1-5 minutes, about 2-4 minutes or preferably about 3 minutes.
  • the invention concerns the method, wherein each wash is carried out for about 0.1 minute - 2 minutes, 0.5 minutes - 1.5 minutes or preferably about 1 minute.
  • the invention concerns the method, wherein said wash buffer is phosphate-buffered saline.
  • the invention concerns the method, wherein said wash buffer is 0.01M phosphate-buffered saline-0,05 % polyoxyethylene sorbitan monolaurate.
  • the invention concerns the method, wherein said enzyme blocking substance is a peroxidase block.
  • said invention concerns the method, wherein said peroxidase block is 3% H 2 O 2 in d2H20.
  • the invention concerns the method, wherein said protein blocking substance is casein solution.
  • said invention concerns the method, wherein said enzyme blocking is carried out for 1-60 minutes, 2-55 minutes, 3-50 minutes, 4-45 minutes, 5-40 minutes, 6-35 minutes, 7-30 minutes, 8-25 minutes, 9- 20 minutes 10-15 minutes or about 10 minutes.
  • said invention concerns the method, wherein said protein blocking is carried out for 1-60 minutes, 5-55 minutes, 10-50 minutes, 15-45 minutes, 20-40 minutes, 25-35 minutes, or about 30 minutes.
  • the invention concerns the method, wherein said method further comprises a step of incubation with a reagent that amplify the signal of the primary antibody
  • a reagent that amplify the signal of the primary antibody may be EnvisionTM Flex + Mouse (linker).
  • the invention concerns the method, wherein said reagent that amplify the signal of the primary antibody is an antibody polymer. According to an embodiment, the invention concerns the method, wherein said incubation with a reagent that amplify the signal of the primary antibody is carried out for about 1 - 30 minutes, about 5 - 25 minutes, about 10 - 20 minutes or preferably about 15 minutes.
  • the invention concerns the method, wherein said reagent that amplify the signal of the primary antibody, enhance the signal at least 2 times, at least 3 times, at least 4 times or preferably at least 5 times.
  • the invention concerns the method, wherein said binding with a primary antibody is carried out for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
  • the invention concerns the method, wherein said binding with a primary antibody is carried out for 1 - 240 minutes, 30 - 110 minutes, 60 - 180 minutes,
  • the invention concerns the method, wherein said binding with a primary antibody is carried out for about 120 minutes or about 24 hours. According to an embodiment, the invention concerns the method, wherein said binding with a primary antibody is carried out for a period of time allowing at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99% of the antigens to be bound by the primary antibody. According to an embodiment, the invention concerns the method, wherein said primary antibody is any of the previously mentioned antibodies or an IgGl antibody.
  • the invention concerns the method, wherein said IgGl antibody is a mouse IgGl antibody.
  • the invention concerns the method, wherein said primary antibody has a concentration of 1 - 60 mg/ml, 5 - 55 mg/ml, 10 - 50 mg/ml, 15 - 45 mg/ml, 20 - 40 mg/ml, 25 - 35 mg/ml or about 30 mg/ml.
  • the invention concerns the method, wherein said primary antibody has a concentration of 1-40 mg/ml, 3-35 mg/ml, 10-30 mg/ml, 15-25 mg/ml or preferably about 20 mg/ml.
  • the invention concerns the method, wherein said secondary antibody is conjugated to a labeled polymer.
  • the invention concerns the method, wherein said labeled polymer is a horseradish peroxidase labeled polymer.
  • the invention concerns the method, wherein said secondary antibody is an anti-mouse antibody.
  • the invention concerns the method, wherein said incubation with a secondary antibody is carried out for 1 - 120 minutes, 10 - 110 minutes, 20 - 100 minutes, 30- 90 minutes, 40 - 80 minutes, 50 - 70 minutes or about 60 minutes.
  • the invention concerns the method, wherein said incubation with a secondary antibody is carried out for 1 - 240 minutes, 10 - 230 minutes, 20 - 220 minutes, 30 - 210 minutes, 40 - 200 minutes, 50 - 190 minutes, 60 - 180 minutes, 70 - 170 minutes, 80 - 160 minutes, 90 - 150 minutes, 100 - 140 minutes, 110 - 130 minutes or preferably about 120 minutes.
  • said staining agent is a benzidine.
  • the invention concerns the method, wherein said benzidine is 3,3'-diaminobenzidine.
  • the invention concerns the method, wherein said incubating with a staining agent is carried out for 1-6 minutes, 1,5 -5,5 minutes, 2-5 minutes, 2,5 - 4,5 minutes, 3 -4 minutes or about 3 minutes.
  • the invention concerns the method, wherein said incubating with a staining agent is carried out for about 1 - 10 minutes, about 1 - 9 minutes, about 2 - 7 minutes or preferably about 3 - 5 minutes.
  • the invention concerns the method, comprising a step, wherein said tissue sample is counterstained with a counterstaining agent.
  • a counterstaining agent is a substance used to provide contrast so the antibody-stained cells stand out more than it does without said counterstaining agent.
  • the invention concerns the method, wherein said counterstaining agent is hematoxylin and optionally eosin.
  • the invention concerns the method, wherein said sample is dehydrated after counterstaining.
  • the invention concerns the method, wherein said tissue sample is coverslipped after dehydration.
  • the invention concerns the method, wherein one or more or all of said steps are separated by at least one wash according to the invention.
  • the invention concerns the method, wherein said steps are performed in the following order:
  • Blocking of enzymes with an enzyme blocking substance ii. Blocking of proteins with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
  • the invention concerns the method, wherein the preparation of said tissue sample comprises one or more or all of the following steps:
  • the invention concerns the method, wherein the method comprises one or more or all of the following steps:
  • the invention concerns the method, wherein said paraffin embedded sample is microtome-sectioned to a thickness of 1 to 7 mm thick, 2 to 6 mm thick or about 3 to 5 mm thickness.
  • the invention concerns the method, wherein said tissue sample is slide-mounted.
  • the invention concerns the method, wherein the preparation for staining and staining comprises one or more or all of the following steps:
  • the invention concerns the method, wherein said sample has formerly been embedded in paraffin. According to an embodiment, the invention concerns the method, wherein said sample has been or is embedded in paraffin and/or formalin fixed.
  • the invention concerns the method, wherein said sample is deparaffinized. According to an embodiment, the invention concerns the method, wherein said antigen retrieval is carried out by a buffer.
  • the invention concerns the method, wherein said buffer is Tris-EDTA buffer.
  • EDTA is also referred to as ethylenediaminetetraacetic acid.
  • the invention concerns the method, wherein said Tris-EDTA buffer has a pH value of 7 - 10, 7.5 - 9.5, 8 - 9 or about 9.
  • the invention concerns the method, wherein said Tris-EDTA buffer is 60 - 140 °C, 65 - 135 °C, 70 - 130 °C, 75 - 125 °C, 80 - 120 °C, 85 - 115 °C, 90 - 110 °C, 95 - 105 °C or about 100 °C.
  • the invention concerns the method, wherein said antigen retrieval is carried out for 1-60 minutes, 2-55 minutes, 3-50 minutes, 4-45 minutes, 5-40 minutes, 6-35 minutes, 7-30 minutes, 8-25 minutes, 9- 20 minutes, 10-15 minutes or about 15 minutes.
  • the invention concerns the method, wherein said steps are performed at 0 - 8 °C, 0.5 - 7.5 °C, 1-7 °C, 1.5 - 6.5 °C, 2-6 °C, 2.5 -5.5 °C, 3 - 5 °C, 3.5 - 4.5 °C or about 4 °C.
  • the invention concerns the method, wherein said steps are performed for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours. According to an embodiment, the invention concerns the method, wherein the steps are followed by one or more or all of the steps described above.
  • the invention concerns the method, wherein said steps are performed in the following order:
  • the invention concerns the method, wherein the steps are performed in the following order: Blocking, binding, incubation, staining.
  • the invention concerns the method, wherein the preparation of said tissue sample comprises one or more or all of the following steps:
  • tissue sample is a frozen sample or a thawed sample.
  • the invention concerns the method, wherein said tissue sample is a thawed sample.
  • the invention concerns the method, wherein said optimal cutting temperature medium comprises one or more or all of the following: polyvinyl alcohol, polyethylene glycol or non-reactive ingredients.
  • the invention concerns the method, wherein said sample is maintained below -30 °C, - 35 °C, -40 °C, -45 °C, -50 °C, -55 °C, -60 °C, -65 °C, -70 °C, -75 °C, - 80 °C, -85 °C, -90 °C, -95 °C or below -100 °C
  • the invention concerns the method, wherein said sample is sectioned to a thickness of 1-10 mm thick, 1.5 - 9.5 mm thick, 2 - 9 mm thick, 2.5 - 8.5 mm thick, 3 - 8 mm thick, 3.5 - 7.5 mm thick, 4 - 7 mm thick, 4.5 - 6.5 mm thick, 5 - 6 mm thick or about 5 mm thicknesses.
  • the invention concerns the method, wherein said sample is sectioned using cryostat. According to an embodiment, the invention concerns the method, wherein said sample is transferred onto a charged slide.
  • the invention concerns the method, wherein said sample is air-dried. According to an embodiment, the invention concerns the method, wherein said sample is air-dried at room temperature, wherein said room temperature is 10 °C - 40 °C.
  • the invention concerns the method, wherein said room temperature is selected among 10 - 40 °C, 15 - 35 °C, 17 - 33 °C, 18 - 30 °C, 19 - 27 °C or about 20 - 25 °C.
  • the invention concerns the method, wherein said sample is air-dried for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
  • the invention concerns the method, wherein the method comprises one or more or all of the following steps: B.l Exposure of a tissue sample to room temperature, wherein said room temperature is 10 - 40 °C.
  • the invention concerns the method, wherein said room temperature is selected among 10 - 40 °C, 15 - 35 °C, 17 - 33 °C, 18 - 30 °C, 19 - 27 °C or about 20 - 25 °C.
  • the invention concerns the method, wherein said exposure to room temperature is carried out for 1 - 60 minutes, 5 - 55 minutes, 10 - 50 minutes, 15 - 45 minutes, 20 - 40 minutes, 25 - 35 minutes or about 30 minutes.
  • the invention concerns the method, wherein said sample is fixated in paraformaldehyde.
  • the invention concerns the method, wherein said sample is fixated in acetone.
  • the invention concerns the method, wherein said acetone is - 10 - 20 °C, -5 - 15 °C, 0 - 10 °C, 0.5 - 7 °C, 1 - 6 °C or 2 - 5 °C.
  • the invention concerns the method, wherein said fixation is carried out for 1-20 minutes, 5 - 15 minutes or about 10 minutes.
  • the invention concerns the method, wherein said steps are followed by one or more or all of the steps previously described.
  • the invention concerns the method, wherein said cancer cells are cancer cells from any cancer type that express or may express B7-H3.
  • the invention concerns the method, wherein said cancer cells are selected among carcinoma, sarcoma, lymphoma and leukemia cancer cells.
  • the invention concerns the method, wherein said cancer cells are selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or gastrointestinal cancer cells.
  • said cancer cells are selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or gastrointestinal cancer cells.
  • the invention concerns the method, wherein said cancer cells are ovarian cancer cells.
  • the invention concerns the method, wherein said ovarian cancer cells are adenocarcinoma cells.
  • the invention concerns the method, wherein said adenocarcinoma cells are serous adenocarcinoma cells.
  • the invention concerns the method, wherein said adenocarcinoma cells are serous papillary adenocarcinoma cells.
  • the invention concerns the method, wherein said cancer cells are gastric cancer cells.
  • the invention concerns the method, wherein said gastric cancer cells are gastric carcinoma cells.
  • the invention concerns the method, wherein said gastric carcinoma cells are gastric adenocarcinoma cells. According to an embodiment, the invention concerns the method, wherein said gastric carcinoma cells are gastric cystadenocarcinoma cells.
  • the invention concerns the method, wherein said cancer cells are neuroblastoma cancer cells. According to an embodiment, the invention concerns the method, wherein said cancer cells are from a metastasis, a high-risk cancer and/or a recurrent cancer.
  • the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample:
  • Binding with a primary antibody wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1, and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 30ug/ml, and wherein said exposure is to be carried out for about 24 hours, and wherein said exposure is to be carried out in about in 4°C ix.
  • EDTA is also referred to as ethylenediaminetetraacetic acid.
  • PBS is also referred to as phosphate-buffered saline.
  • CAS-protein block is also referred to as casein solution.
  • DAB is also referred to as 3,3'-diaminobenzidine.
  • d2H20 is also referred to as double-distilled water.
  • the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Remove the frozen sample from -80 °C and keep in room temperature, wherein said room temperature is selected among any of the temperatures described above as room temperatur 110, and wherein said samples are kept in room temperature for about 30 minutes ii.
  • the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and wherein said antigen retrieval is carried out for about 20 minutes iii. Cool down for about 20 minutes iv. First rinse in d2H20 for about 1 minute v. Second rinse in d2H20 for about 1 minute vi. First wash with 0.01M phosphate-buffered saline for about 2 minutes vii. Second wash with 0.01M phosphate-buffered saline for about 2 minutes viii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes ix.
  • Blocking enzymes with peroxidase block wherein said peroxidase block is 3% H 2 O 2 in d2H20, and wherein said exposure is carried out for about 10 minutes x.
  • the invention concerns the method, wherein said tissue sample is an ovarian tissue sample.
  • the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and wherein said antigen retrieval is carried out for about 20 minutes iii. Cool down for about 20 minutes iv. First rinse in d2H20 for about 1 minute v. Second rinse in d2H20 for about 1 minute vi. First wash with 0.01M phosphate-buffered saline for about 2 minutes vii. Second wash with 0.01M phosphate-buffered saline for about 2 minutes viii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes ix.
  • Blocking enzymes with peroxidase block wherein said peroxidase block is 3% H 2 O 2 in d2H20, and wherein said exposure is carried out for about 10 minutes x.
  • the invention concerns the method, wherein said tissue sample is a gastric tissue sample.
  • the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
  • the invention concerns a method for detecting the clinical benefit of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
  • the individual is a specific individual or a specific cancer patient, as the method is to be used for each individual patient.
  • the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer, wherein the method is performed on tissue samples comprising cancer cells.
  • the invention concerns a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof, wherein said chimeric antigen receptor is used according to a method of the invention.
  • the invention concerns a CAR-T cell expressing the chimeric antigen receptor.
  • the invention concerns a population of CAR-T cells.
  • the invention concerns a composition comprising the population of CAR-T cells.
  • the invention concerns a method of treating cancer in an individual, wherein the method comprises use of a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
  • the invention concerns a method for treating cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the indication, such as ovarian cancer, for treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
  • the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
  • the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
  • the invention concerns the method, wherein said tissue sample is a tissue sample from said individual.
  • the invention concerns the method, wherein said method evaluates overexpression of B7-H3.
  • the invention concerns the method, wherein said cancer cells are selected among any of the cancer cells as described above.
  • the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is any of the antibodies or antigen-binding fragments as described above.
  • the invention concerns the method, wherein said cancer is any cancer type that express or may express B7-H3.
  • the invention concerns the method, wherein said cancer is selected among a carcinoma, a sarcoma, a lymphoma and a leukemia.
  • the invention concerns the method, wherein said cancer is selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or a gastrointestinal cancer
  • said cancer is selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or a gastrointestinal cancer
  • the invention concerns the method, wherein said cancer is an ovarian cancer.
  • the invention concerns the method, wherein said ovarian cancer is an adenocarcinoma.
  • the invention concerns the method, wherein said adenocarcinoma is a serous adenocarcinoma. According to an embodiment, the invention concerns the method, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
  • the invention concerns the method, wherein said cancer is a gastric cancer.
  • the invention concerns the method, wherein said gastric cancer is a carcinoma.
  • the invention concerns the method, wherein said carcinoma is an adenocarcinoma.
  • the invention concerns the method, wherein said carcinoma is a cystadenocarcinoma.
  • the invention concerns the method, wherein said cancer is neuroblastoma.
  • the invention concerns the method, wherein said cancer is a metastasis, a high-risk cancer and/or a recurrent cancer.
  • the invention concerns the method, wherein said treatment is combined with chemotherapy and/or surgery.
  • the invention concerns a kit of parts, adapted to carry out a method of the invention.
  • the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is to be used in the treatment of a cancer.
  • the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify the clinical benefit a patient has or will have from treatment with an antigen or antigen-binding fragment thereof, and wherein said patient is a cancer patient.
  • the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer.
  • the invention concerns a kit of parts, wherein said kit is used in detecting B7-H3 expression in a tissue sample comprising cancer cells, and wherein said sample is selected among a sample that have been embedded in paraffin and a sample have been frozen.
  • the invention concerns the kit, comprising an anti-B7-H3 antibody or antigen-binding fragment thereof used to detect B7-H3 expression.
  • the invention concerns the kit, wherein said kit comprises a primary antibody.
  • the invention concerns the kit, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof, or said primary antibody, is any of the antibodies or antigen-binding fragments thereof as described above.
  • the invention concerns the kit, wherein said kit uses antibodies to detect proteins in cells of said tissue sample.
  • the invention concerns the kit, wherein said kit comprises a secondary antibody.
  • the invention concerns the kit, wherein said secondary antibody is an anti-mouse antibody.
  • the invention concerns the kit, wherein said secondary antibody is conjugated to an enzyme.
  • the invention concerns the kit, wherein said enzyme is a peroxidase.
  • the invention concerns the kit, wherein said peroxidase is Horseradish peroxidase.
  • the invention concerns the kit, wherein said kit comprises an enzyme block.
  • the invention concerns the kit, wherein said enzyme block is a peroxidase block.
  • the invention concerns the kit, where said peroxidase block is 3% H 2 O 2 in d2H20.
  • the invention concerns the kit, wherein said kit comprises a protein block.
  • the invention concerns the kit, wherein said protein block is casein solution.
  • the invention concerns the kit, wherein said kit comprises a substance for chromogenic staining.
  • the invention concerns the kit, wherein said substance for chromogenic staining is a benzidine.
  • the invention concerns the kit, wherein said benzidine is 3,3'- Diaminobenzidine.
  • the invention concerns the kit, wherein said substance for chromogenic staining is a counterstaining agent.
  • the invention concerns the kit, wherein said counterstaining agent is hematoxylin and optionally eosin.
  • the invention concerns the kit, wherein said kit further comprises a reagent that amplify the signal of the primary antibody.
  • the invention concerns the kit, wherein said reagent that amplify the signal of the primary antibody is an antibody polymer.
  • the invention concerns the kit, wherein said kit comprises a substance used to wash.
  • the invention concerns the kit, wherein said substance used to wash is a wash buffer.
  • the invention concerns the kit, wherein said wash buffer is phosphate-buffered saline.
  • the invention concerns the kit, wherein said wash buffer is 0,01M phosphate-buffered saline-0,05 % polyoxyethylene sorbitan monolaurate.
  • the invention concerns the kit, wherein the kit further comprises water.
  • the invention concerns the kit, wherein said water is d2H20.
  • the invention concerns the kit, wherein said cancer cells are selected among any of the cancer cells as described above.
  • the invention concerns the kit, wherein said cancer is selected among any of the cancers as described above.
  • the invention concerns a method for treating cancer in an individual, wherein the method comprises use of a kit according to the invention.
  • the invention concerns the method, wherein said cancer is selected among any of the cancers as described above. According to an embodiment, the invention concerns the method, wherein said cancer is a gastric cancer.
  • the invention concerns the method, wherein said gastric cancer is a carcinoma.
  • the invention concerns the method, wherein said carcinoma is an adenocarcinoma.
  • the invention concerns the method, wherein said carcinoma is a cystadenocarcinoma.
  • the invention concerns the method, wherein said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
  • said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
  • the invention concerns the method, wherein said cancer is an ovarian cancer.
  • the invention concerns the method, wherein said ovarian cancer is an adenocarcinoma.
  • the invention concerns the method, wherein said adenocarcinoma is a serous adenocarcinoma.
  • the invention concerns the method, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
  • the invention concerns the method, wherein said ovarian cancer is selected from the group consisting of epithelial carcinoma, serous carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed mijllerian tumor (carcinosarcoma), mucinous tumors, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial cancers, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor/endodermal sinus tumor, embryonal carcinoma, polye
  • the invention concerns the method, wherein said cancer is a neuroblastoma.
  • the invention concerns the method, wherein the distribution of cells is selected from the group consisting of diffuse and multifocal, preferably as determined by the methods of Example 2.
  • the invention concerns the method, wherein the incidence is 1, preferably 2, more preferred 3, preferably 4, preferably as determined by the methods of Example 2.
  • the invention concerns the method, wherein the incidence is 2-4, preferably 3-4, preferably as determined by the methods of Example 2.
  • the invention concerns the method, wherein the staining intensity is 1, preferably 2, more preferred 3, preferably 4, preferably as determined by the methods of Example 2.
  • the invention concerns the method, wherein the staining intensity is 2-4, preferably 3-4, preferably as determined by the methods of Example 2.
  • the invention concerns the method, wherein the staining is granular, preferably as determined by the methods of Example 2. According to an embodiment, the invention concerns the method, wherein the staining is located on the membrane or cytoplasmic, preferably as determined by the methods of Example 2.
  • the invention concerns the method, wherein the staining is located on the membrane, preferably as determined by the methods of Example 2.
  • the invention concerns the method, wherein the subcellular location is selected from the group consisting of Granular (G), Membrane (M), Cytoplasmic (C), Extracellular (E), Vasculature (V), Intranuclear (In), Apical (A) and Basal (B), preferably as determined by the methods of Example 3.
  • the invention concerns the method, wherein the amount of positive tumor cells in percentage is selected from the group consisting of up to 29%, 30- 49%, 50-74%, and 75-100%, preferably as determined by the methods of Example 3.
  • the invention concerns the method, wherein the amount of positive tumor cells in percentage is above 10%, above 20%, preferably above 30%, preferably as determined by the methods of Example 3.
  • the invention concerns the method, wherein the incidence of positive tumor cells is selected from the group consisting of normal, minimal, mild, moderate and severe, preferably as determined by the methods of Example 3.
  • the invention concerns the method, wherein the intensity of positive staining at cellular level is selected from the group consisting of equivocal, mild (weak), moderate, strong and intense, preferably as determined by the methods of Example 3.
  • the invention concerns the method, wherein the incidence of staining represents a mild to severe distribution of positive tumor cells, preferably as determined by the methods of Example 3.
  • the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
  • the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
  • the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No.
  • a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antigen-binding fragment thereof is a single chain variable fragment (scFv).
  • scFv single chain variable fragment
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said scFv comprises a portion of the amino acid sequence set forth in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said scFv comprises at least one of the amino acid substitutions K13E, R18Q, R45Q, K103E and K107E.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof is a murine 8H9 antibody or an antigen binding fragment thereof. According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is humanized.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is radiolabeled.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said radiolabel is selected among a PET label and a SPECT label.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said PET label is selected among 124 l, 225 Ac and 89 Zr.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said SPECT label is selected among 131 l, 177 Lu, 99 mTc, 64 Cu and 89 Zr.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is conjugated to a chelator compound.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said chelator compound is bound to a radioactive isotope.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said radioactive isotope is selected among 124 l, 131 l and 177 Lu or 99 mTc, 64 Cu (chelated to NOTA) and 89 Zr (chelated to DFO).
  • said radioactive isotope is selected among 124 l, 131 l and 177 Lu or 99 mTc, 64 Cu (chelated to NOTA) and 89 Zr (chelated to DFO).
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said DOTA is a variant of DOTA.
  • the invention concerns an antibody or antigen binding fragment according to the invention, wherein said DTPA is a variant of DTPA.
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said ovarian cancer is an adenocarcinoma.
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said adenocarcinoma is a serous adenocarcinoma.
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer
  • said ovarian cancer is selected from the group consisting of epithelial carcinoma, serous carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed müllerian tumor (carcinosarcoma), mucinous tumors, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial cancers, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said gastric cancer is a carcinoma.
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said carcinoma is an adenocarcinoma.
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said carcinoma is a cystadenocarcinoma.
  • the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
  • gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
  • Fig. 1 provides a flow chart of a method for detection related to an embodiment of the invention.
  • Fig. 2A and 2B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 30 mg/mL, overnight at 4 °C. There is severe granular cell surface and intracellular staining of tumor cells.
  • Fig. 2A shows a field of view with 20 times magnification
  • Fig. 2B shows a field of view with 40 times magnification.
  • the histopathologic score was 4/4.
  • Fig. 3A and 3B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with isotype control mouse IgG1 antibody ( MslgG1). The slides were stained with 30 mg/mL MslgG1 overnight at 4 °C. There is no staining of tumor cells.
  • Fig. 3A shows a field of view with 20 times magnification and Fig. 2B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 4A and 4B show the immunohistochemistry of FFPE sections of neuroblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 30 mg/mL, overnight at 4 °C. There is strong to intense granular cell surface and some intracellular staining of tumor cells.
  • Fig. 4A shows a field of view with 20 times magnification
  • Fig. 4B shows a field of view with 40 times magnification.
  • the histopathologic score was 4/3.
  • Fig. 5A and 5B show the immunohistochemistry of FFPE sections of neuroblastoma with MslgG1. The slides were stained with 30 mg/mL MslgG1 overnight at 4 °C. There is no staining.
  • Fig. 5A shows a field of view with 20 times magnification and Fig. 5B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 6A and 6B show the immunohistochemistry of FFPE sections of medulloblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • Fig. 6A shows a field of view with 20 times magnification
  • Fig. 6B shows a field of view with 40 times magnification.
  • the histopathologic score was 4/4.
  • Fig. 7A and 7B show the immunohistochemistry of FFPE sections of medulloblastoma with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining.
  • Fig. 7A shows a field of view with 20 times magnification and Fig. 7B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 8A and 8B show the immunohistochemistry of FFPE sections of glioblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is intense granular cell surface and intracellular staining of tumor cells.
  • Fig. 8A shows a field of view with 20 times magnification
  • Fig. 8B shows a field of view with 40 times magnification.
  • the histopathologic score was 4/4.
  • Fig. 9A and 9B show the immunohistochemistry of FFPE sections of glioblastoma with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining.
  • Fig. 9A shows a field of view with 20 times magnification and Fig. 9B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 10A and 10B show the immunohistochemistry of FFPE sections of ovarian cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is moderate multifocal granular cell surface and intracellular and vascular staining of a subset of tumor cells.
  • Fig. 10A shows a field of view with 20 times magnification
  • Fig. 10B shows a field of view with 40 times magnification.
  • the histopathologic score was 2/2.
  • Fig. 11A and 11B show the immunohistochemistry of FFPE sections of ovarian cancer with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining.
  • Fig. 11A shows a field of view with 20 times magnification and Fig. 11B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 12A and 12B show the immunohistochemistry of FFPE sections of sarcoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is moderate multifocal granular cell surface and intracellular staining of a subset of tumor cells.
  • Fig. 12A shows a field of view with 20 times magnification
  • Fig. 12B shows a field of view with 40 times magnification.
  • the histopathologic score was 2/2.
  • Fig. 13A and 13B show the immunohistochemistry of FFPE sections of sarcoma with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining. Fig.
  • FIG. 13A shows a field of view with 20 times magnification and Fig. 13B shows a field of view with 40 times magnification.
  • the histopathologic score was 0/0.
  • Fig. 14A and 14B show the immunohistochemistry of FFPE sections of melanoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is severe granular cell surface and intracellular staining of tumor cells.
  • Fig. 14A shows a field of view with 20 times magnification
  • Fig. 14B shows a field of view with 40 times magnification.
  • the histopathologic score was 4/4.
  • Fig. 15A and 15B show the immunohistochemistry of FFPE sections of melanoma with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining of tumor cells. It shows moderate staining of macrophages.
  • Fig. 15A shows a field of view with 20 times magnification and Fig. 15B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 16A and 16B show the immunohistochemistry of FFPE sections of liver cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells.
  • Fig. 16A shows a field of view with 20 times magnification
  • Fig. 16B shows a field of view with 40 times magnification.
  • the histopathologic score was 3/3.
  • Fig. 17A and 17B show the immunohistochemistry of FFPE sections of normal liver with an antibody comprising a heavy chain according to SEQ ID No.
  • Fig. 17A shows a field of view with 20 times magnification
  • Fig. 17B shows a field of view with 40 times magnification.
  • the histopathologic score was 0/0.
  • Fig. 18A shows the immunohistochemistry of frozen sections of non-small cell lung carcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 2 ⁇ g/mL. There is a moderate number of strong positive stained tumor cells.
  • Fig. 18A shows a field of view with 40 times magnification. The histopathologic score was 2/3.
  • Fig. 18B shows the immunohistochemistry of frozen sections of non-small cell lung carcinoma with MslgG1. The slides were stained with 2 ⁇ g/mL MslgG1. There is no staining. Fig. 18B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 19A shows the immunohistochemistry of frozen sections of lung adenocarcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 2 ⁇ g/mL. There is a strong positive stained subset of tumor cells.
  • Fig. 19A shows a field of view with 40 times magnification. The histopathologic score was 3/3.
  • Fig. 19B shows the immunohistochemistry of frozen sections of lung adenocarcinoma with MslgG1.
  • the slides were stained with 2 ⁇ g/mL MslgG1. There is no staining.
  • Fig. 19B shows a field of view with 40 times magnification.
  • the histopathologic score was 0/0.
  • Fig. 20A shows the immunohistochemistry of frozen sections of melanoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 2 ⁇ g/mL. There are strong multifocal stained tumor cells.
  • Fig. 20A shows a field of view with 40 times magnification.
  • Fig. 20B shows the immunohistochemistry of frozen sections of melanoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 20B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 21A shows the immunohistochemistry of frozen sections with Sarcoma with an antibody comprising a heavy chain according to SEQ ID no. 1, and a light chain according to SEQ ID no. 2.
  • the slides were stained with said antibody, at a concentration of 2 mg/mL. There is strong to intense stained tumor cells.
  • Fig. 21A shows a field of view with 40 times magnification. The histopathologic score was 4/4.
  • Fig. 21B shows the immunohistochemistry of frozen sections of Sarcoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 21B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 22A shows the immunohistochemistry of frozen sections of ovarian cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 2 mg/mL. There is strong to intense multifocal stained tumor cells.
  • Fig. 22A shows a field of view with 40 times magnification. The histopathologic score was 4/3.
  • Fig. 22B shows the immunohistochemistry of frozen sections of ovarian cancer with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 22B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 23A shows the immunohistochemistry of frozen sections of glioblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
  • the slides were stained with said antibody, at a concentration of 2 mg/mL. There is a strong stained subset of tumor cells.
  • Fig. 23A shows a field of view with 40 times magnification. The histopathologic score was 3/3.
  • Fig. 23B shows the immunohistochemistry of frozen sections of glioblastoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 23B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
  • Fig. 24A and 24B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is severe granular cell surface and intracellular staining of tumor cells.
  • Fig. 24A shows a field of view with 20 times magnification and Fig. 24B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
  • Fig. 25A and 25B show the immunohistochemistry of FFPE sections of neuroblastoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is severe granular cell surface staining of tumor cells.
  • Fig. 25A shows a field of view with 20 times magnification and Fig. 25B shows a field of view with 40 times magnification. The histopathologic score was 4/3.
  • Fig. 26A and 26B show the immunohistochemistry of FFPE sections of medulloblastoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells.
  • Fig. 26A shows a field of view with 20 times magnification and Fig. 26B shows a field of view with 40 times magnification. The histopathologic score was 3/4.
  • Fig. 27A and 27B show the immunohistochemistry of FFPE sections of glioblastoma with commercially available a nti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells.
  • Fig. 27A shows a field of view with 20 times magnification and Fig. 27B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
  • Fig. 28A and 28B show the immunohistochemistry of FFPE sections of ovarian cancer with commercially available anti-B7-H3 antibody.
  • the slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is moderate granular cell surface, intracellular and vascular staining of tumor cells.
  • Fig. 28A shows a field of view with 20 times magnification and Fig. 28B shows a field of view with 40 times magnification.
  • the histopathologic score was 3/3.
  • Fig. 29A and 29B show the hematoxylin and eosin counter-staining of FFPE section of non- small cell lung carcinoma. Well preserved tumor tissue is shown.
  • Fig. 29A shows a field of view with 20 times magnification and
  • Fig. 29B shows a field of view with 40 times magnification.
  • Fig. 30A and 30B show the hematoxylin and eosin counter-staining of FFPE section of neuroblastoma.
  • Well preserved tumor tissue is shown.
  • Fig. 30A shows a field of view with 20 times magnification and
  • Fig. 30B shows a field of view with 40 times magnification.
  • Fig. 31A and 31B show the hematoxylin and eosin counter-staining of FFPE section of medulloblastoma.
  • Well preserved tumor tissue is shown.
  • Fig. 31A shows a field of view with 20 times magnification
  • Fig. 31B shows a field of view with 40 times magnification.
  • Fig. 32A and 32B show the hematoxylin and eosin counter-staining of FFPE section of ovarian cancer. Well preserved tumor tissue is shown.
  • Fig. 32A shows a field of view with 20 times magnification and
  • Fig. 32B shows a field of view with 40 times magnification.
  • Fig. 33A and 33B show the hematoxylin and eosin counter-staining of FFPE section of sarcoma.
  • Well preserved tumor tissue is shown.
  • Fig. 33A shows a field of view with 20 times magnification and
  • Fig. 33B shows a field of view with 40 times magnification.
  • Fig. 34A and 34B show the hematoxylin and eosin counter-staining of FFPE section of sarcoma.
  • Well preserved tumor tissue is shown.
  • Fig. 34A shows a field of view with 20 times magnification
  • Fig. 34B shows a field of view with 40 times magnification.
  • Fig. 35A and 35B show the hematoxylin and eosin counter-staining of FFPE section of glioblastoma. Well preserved tumor tissue is shown.
  • Fig. 35A shows a field of view with 20 times magnification and
  • Fig. 35B shows a field of view with 40 times magnification.
  • Fig. 36-40 shows IHC staining on 25 TMA Ovarian Cancer Cases. Donor ID as in table 7.
  • Fig. 41 shows H&E and IHC staining of patient's gastric tissue (ID: 001, Spindle cell tumor, possible leiomyoma, benign).
  • ID 001, Spindle cell tumor, possible leiomyoma, benign.
  • H&E image shows a large mass in the muscularis layer (A, black star).
  • the tumor consists of spindle cells which showed no immunoreactivity with 8H9 antibody (B and D) or MslgG (C and E).
  • Fig. 42 shows IHC staining of patient's gastric tissue (ID: 001-2, Gastric Adenocarcinoma).
  • ID 001-2, Gastric Adenocarcinoma
  • 8H9 antibody is expressed on the membrane of 80% of tumor cells with grade 3 staining intensity (A). Capillaries are often filled with neutrophils and lymphocytes as well as tumor cells (B, C). At 20 mg/ml, membrane staining with grade 2-3 staining intensity is observed in about 50% of tumor cells (D). The expression of 8H9 antibody is detected on membrane of most tumor cells (E, F). No immunoreactivity is found with MslgG control isotype (Data not shown).
  • Fig. 43A shows H&E and IHC staining of patient's gastric tissue (ID 002; gastric adenocarcinoma).
  • H&E image shows a protruding proliferative mass in the mucosa (A, doted box).
  • the tumor consists of signet ring cells (B, yellow arrows).
  • the immunoreactivity with 8H9 antibody was detected in the membrane and cytoplasm of neoplastic epithelium (B and D). No immunostaining was found in sections treated with MslgG (C and E).
  • Fig. 43B shows at 20 mg/ml, there is occasional weak granular membrane staining on less than 10% of the tumor cells (F and G, red arrow). Vascular endothelial staining was observed in tumor areas (H).
  • Fig. 44A shows H&E and IHC staining of patient's gastric tissue (ID: 003, Gastric Carcinoma).
  • H&E image shows a nodule of tumor with no gastric structure (A, doted circle).
  • the immunoreactivity with 8H9 antibody was detected in about half of all neoplastic epithelium (B) with a diffuse and cytoplasmic pattern (D, yellow arrows). No immunostaining was found in sections treated with MslgG (C and E).
  • Fig. 44B shows at 20 mg/ml, there is little to no positive membrane staining (F and G). Vascular endothelial staining was present in tumor areas (H).
  • Fig. 45A shows H&E and IHC staining of patient's gastric tissue (ID: 004, Gastric Adencarcinoma).
  • H&E image shows a polypoid proliferative mass in the mucosa and submucosa including numerous small lymphoid nodules (A, starts represents lymphoid nodules).
  • the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
  • Fig. 45 B shows at 20 mg/ml, there is moderate granular membrane staining on about 20% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
  • Fig. 46A shows H&E and IHC staining of patient's gastric tissue (ID: 005, Gastric Adenocarcinoma).
  • H&E image shows a large diffuse mass in the muscularis and serosa with severe inflammation, congestion, and hemorrhage (A).
  • the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
  • Fig. 46B shows At 20 mg/ml, there is moderate granular membrane staining on about 30% of the tumor cells in areas of less differentiated tumor cells (F and G, red arrows).
  • Fig. 47A shows H&E and IHC staining of patient's gastric tissue (ID: 006, Gastric Adenocarcinoma).
  • H&E image shows a large diffuse infiltrative mass in the mucosa infiltrating the submucosa, muscularis, and serosa and serosa (A).
  • the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
  • Fig. 47B shows at 20 ug/ml, there is moderate granular membrane staining on about 30% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
  • Fig. 48A shows H&E and IHC staining of patient's gastric tissue (ID: 007, Gastric Cystadenocarcinoma).
  • H&E image shows a large diffuse infiltrative mass invading mucosa, submucosa, muscularis, and serosa (A).
  • the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
  • Fig. 48B shows at 20 ug/ml, there is occasional granular membrane staining on less than 10% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
  • Fig. 49A shows H&E and IHC staining of patient's gastric tissue (ID: 008, Gastric Adenocarcinoma).
  • H&E image shows a full thickness of gastric mucosa with a protruding proliferative mass and expansion of submucosa (A).
  • the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
  • Fig. 49B shows at 20 mg/ml, the majority of tumor cells demonstrated granular membrane staining (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
  • Fig. 50A shows H&E and IHC staining of patient's gastric tissue (ID: 009, Gastric Adenocarcinoma).
  • H&E image shows normal and neoplastic gastric tissues (A).
  • the immunoreactivity with 8H9 antibody was detected in about 50% of neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
  • Fig. 50B shows At 20 mg/ml, there is weak granular membrane staining on about 30% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
  • Immunohistochemical staining is in general a challenging assay and the selection of an optimal condition requires manipulating a variety of parameters.
  • the following conditions did not provide optimal conditions for immunohistochemical staining of formalin fixed paraffin embedded (FFPE) samples with murine 8H9 antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2:
  • Carcinoma Ovarian Cancer, Medulloblastoma, Neuroblastoma, Glioblastoma, Sarcoma and Melanoma.
  • the preset study has determined whether a murine 8H9 antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2 targeting human immunoregulatory B7-H3 protein, binds to any human tumor tissues.
  • the present study has assessed potential binding of a murine 8H9 antibody to neuroblastoma, medulloblastoma, ovarian cancer, non-small cell lung cancer (NSCLS), sarcoma, melanoma and glioblastoma.
  • NSCS non-small cell lung cancer
  • sarcoma melanoma
  • glioblastoma Specific positive tissue staining results suggest cancer types that may be targeted by the antibody in vivo in humans.
  • the Isotype Control Mouse lgGl binds to the immunogen: TNP (Trinitrophenol) + KLH (keyhole limpet haemocyanin).
  • All tissues were gross trimmed, processed through a graded series of alcohols, oriented and embedded in paraffin, microtome-sectioned at 3 to 5 mm thicknesses, slide-mounted, immunohistochemically stained with mu8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2, and control antibodies, and cover-slipped by standard methodology.
  • the unfixed frozen tissues were embedded in optimal cutting temperature medium (OCT) and blocks were prepared.
  • OCT tissue blocks were placed in sealed plastic bags or other appropriate containers, and maintained below -70 °C pending sectioning.
  • Sections were stained with mu8H9 antibody comprising a heavy chain sequence according to SEQ ID No.l and a light chain sequence according to SEQ ID No. 2 in order to assess expression of the B7-H3 in these tissues. Staining were performed on both formalin fixed paraffin embedded sections and frozen sections.
  • the staining consisted of the following steps, separated by rinses with wash buffer: peroxidase block for 10 minutes; CAS block (casein solution) for 30 minutes; primary antibody overnight; horseradish peroxidase labeled polymer conjugated to an anti-mouse antibody for 60 minutes; 3,3'-diaminobenzidine for 3 minutes.
  • the slides then were counterstained with hematoxylin, dehydrated, and coverslipped by standard methodologies.
  • 0 Normal - Tissue considered to be normal under the conditions of the study and considering the age, sex and strain of the animal concerned. Alterations may be present which, under other circumstances, would be considered deviations from normal.
  • the pathologist examined all of the submitted tissue sections using light microscopy and recorded the findings directly into tables. The data generated was used by the pathologist in assessment of the histopathologic findings associated with epitope expression. Results
  • Mu8H9 staining was detected diffusely and multifocally in a subset of tumor cells in all tissues. The staining was granular, generally strong to intense (Grade 3-4) and located on the membrane and occasionally cytoplasmic, although there was some vasculature and intracellular staining as well. The staining of most of the tumor tissues was subjected to immunohistochemical staining with commercially available anti-BH-73 antibody. The condition of staining and staining pattern was similar to that of mu8H9, in particular for the IHC staining of frozen sections.
  • Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 recognized the target molecule B7-H3 in FFPE samples of different tumor types under following condition: - Antigen retrieval Tris-EDTA pH 9 at 100°C (without any pressure which avoided tissue damage).
  • Mu8H9 comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 stained the tumor cells in all tumor tissues diffusely and in some cases multifocally.
  • the staining pattern was generally intense, granular and located on the membrane, although there was cytoplasmic, intracellular, and vascular staining as well. Positive staining was evident in all tumor types, however appeared to be more extensive in the FFPE sections of non-small cell carcinoma, neuroblastoma, medulloblastoma, glioblastoma, melanoma and liver cancer. Staining was weak to moderate in ovarian cancer and sarcoma. There was strong positive staining in all frozen tumors, with the exception of moderate staining of lung adenocarcinoma.
  • the objective of this study was to determine whether Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 binds to formalin fixed paraffin embedded ovarian cancer tissues using immunohistochemical techniques. Specific staining was detected diffusely and multifocally in the tumor cells in a majority of the ovarian cancer tissues. Generally, 80% of tumor samples showed positive staining for the antibody representing the expression of B7-H3 in 40-100% of tumor cells. The staining was granular, generally mild to intense (Grade 1-4) with cytoplasmic/membranous pattern. The positive membrane staining was found in majority of tumor cells (50-100%) with overall intensity of +2 to +4.
  • the study protocol from example 2 was used for IHC staining, however, additional steps were taken to optimize the intensity of the signal in the ovarian cancer tissues as described in the section Immunohistochemical Staining Methodology. Initially the staining protocol was optimized for three concentrations of Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 (10, 20 and 30 mg/ml) and the concentration of 20 mg/ml was selected as the optimal concentration for the rest of the study. Von Willebrand Factor (vWF) was used as a marker for quality control of the testing system. The modification and reproducibility of the staining protocol was validated through eight validation runs using the experimental design presented in table 4.
  • vWF Von Willebrand Factor
  • ovarian normal and cancer FFPF tissues from individual donors were microtome- sectioned at 5 mm thicknesses and mounted onto charged slides.
  • Commercially available ovarian cancer at 5 tissue microarray (TMA) slides of 25 cases (50 cores, 25 cores for testing Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 and 25 cores for testing Ms IgG) were used to validate the results of the staining in a large number of tissue samples.
  • Sections were stained with murine 8H9 antibody in order to assess expression of the B7-H3 in these tissues.
  • FFPE tissue sections on slides were deparaffinized, hydrated, and subjected to heated Tris-EDTA buffer (pH9, 100 °C) antigen retrieval. After PBS wash, sections were treated with peroxide block solution for 10 minutes, washed with PBS, and incubated with protein blocking buffer for 30 min at room temperature (RT). Then, the tissues were incubated with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 overnight.
  • the second day of staining was conducted with a modified version of the staining protocol used in Example 2 where the sections were treated with a signal enhancer (EnvisionTM FLEX + Mouse (linker)) for 15 minutes prior to adding HRP conjugated polymer for 60 minutes. After three runs of PBS wash, the sections were exposed to a chromogen reagens (EnVisionTM FLEX DAB+ Chromogen) for 1-2 minutes and the intensity of the signal was evaluated under microscope. The slides then were counterstained with hematoxylin, dehydrated, and coverslipped by standard methodologies. IHC methodology for ovarian FFPE tissue:
  • results of histopathologic scoring with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 are detailed in table 6 and table 7.
  • Table 5 showing the histopathological scoring terms. Representative photomicrographs are shown in Fig. 36-40.
  • the objective of this study was to assess the staining of Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2, a therapeutic agent when radiolabeled as a tool to detect B7-H3, to FFPE ovarian cancer tissue using immunohistochemical techniques. Specific staining was detected diffusely and multifocally in the tumor cells in a majority of the ovarian cancer tissues. The control tissue (normal ovary) dit not show any reaction with 8H9 antibody. The staining was granular, generally mild to intense (Grade 1-4) with cytoplasmic/membranous pattern.
  • Example 4 Histopathological Evaluation of Gastric Cancer Tissues for Expression of B7-H3 (CD276) Using Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
  • the objective of this study was to evaluate histopathologic and immunohistochemical findings of gastric carcinomas from nine patients that were stained with the monoclonal Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
  • This immunohistochemistry study assessed potential binding of the murine 8H9 antibody to human gastric cancer tissues derived from nine patients. Specific positive staining results suggest tumor cell types that may be targeted by 8H9 antibody in vivo in humans.
  • Tissues were then evaluated via light microscopy by a board-certified veterinary pathologist.
  • Sections were stained with murine 8H9 antibody to assess expression of the B7-H3 in these tissues.
  • the staining technique was developed at CBI. The detailed methodology is shown below.
  • PBS (IX) is used as washing buffer for this study.
  • PBS could be replaced by other washing buffer e.g. Tris-EDTA or PBS with Tween-20 (0.05%).
  • Tissues were qualitatively examined by light microscopy by a veterinary pathologist certified by the American College of Veterinary Pathologists. The pathologist prepared a report describing the histologic features and confirming the diagnosis or the purposes of confirming tissue identity. If any numerical semiquantitative assessment was needed, lesions were scored using the industry standard 5-point scoring system as described in example 2 (Mann, et al., 2012). Immunohistochemical Assessment
  • Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 for the gastric cancer tissues in comparison with neuroblastoma (positive control), human heart and liver (negative control) tissues, and commercially available B7-H3, respectively.
  • the specificity of the 8H9 antibody was compared with MslgG control.
  • the sensitivity of the antibody was optimized at 30 mg/ml only for IHC staining uses.
  • M Membrane (M), Nuclear (N), Perinuclear(P) with a granular pattern.
  • H&E Control Characterizing cellular morphology and identifying microstructural abnormality including inflammation, vascular leakage, fibrosis, tumor invasion, ect. Masked assessment for description and diagnosis of patient tissues were applied.
  • H&E Summary histopathologic staining assessment of patient tissues is presented in table 8.
  • Table 9 Summary immunohistochemical staining assessment of patients is presented in Table 9. Representative photomicrographs are presented in Fig. 41-50. Table 9 ICH staining with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 results for each tissue tested
  • Tumor cells were clearly and strongly positive for 8H9 antibody staining. The staining was specific and limited to the tumor cells. Normal cells such as smooth muscle, vessels, nerves, normal mucosa, and vessels were not stained.
  • Results indicate that there is strong binding of 8H9 specifically to the cytoplasm and membrane of the gastric carcinoma tumor cells in the tissues submitted.
  • the leiomyoma (ID 001) was not stained.

Abstract

La présente invention concerne un procédé pour fournir un biomarqueur de l'efficacité et/ou de la sécurité d'un anticorps ou d'un fragment de liaison à l'antigène de celui-ci à l'aide de procédés immunohistochimiques. Les procédés immunohistochimiques précédents faisant appel à des échantillons incorporés dans de la paraffine en combinaison avec un anticorps murin 8H9 n'ont pas permis d'obtenir des résultats satisfaisants. La présente invention concerne un procédé qui peut utiliser des échantillons congelés et des échantillons incorporés dans de la paraffine. En outre, la présente invention concerne un ensemble d'éléments conçu pour mettre en œuvre le procédé de l'invention. De plus, l'invention concerne une méthode de traitement d'un cancer, consistant à mesurer ou à vérifier un biomarqueur de l'efficacité, de l'adéquation et/ou de la sécurité d'un anticorps ou son fragment de liaison à l'antigène, ledit anticorps ou son fragment de liaison à l'antigène étant utilisé pour traiter ledit cancer.
EP20765196.9A 2019-08-30 2020-08-20 Évaluation immunohistochimique de l'expression de b7-h3 Withdrawn EP4022313A1 (fr)

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US7737258B2 (en) 2000-10-18 2010-06-15 Sloan-Kettering Institute For Cancer Research Uses of monoclonal antibody 8H9
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