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WO2025257588A1 - Liant polyspécifique d'antigène de cd16a/tumeur destiné à être utilisé dans le traitement de la résistance à un inhibiteur de point de contrôle immunitaire - Google Patents

Liant polyspécifique d'antigène de cd16a/tumeur destiné à être utilisé dans le traitement de la résistance à un inhibiteur de point de contrôle immunitaire

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Publication number
WO2025257588A1
WO2025257588A1 PCT/IB2024/055675 IB2024055675W WO2025257588A1 WO 2025257588 A1 WO2025257588 A1 WO 2025257588A1 IB 2024055675 W IB2024055675 W IB 2024055675W WO 2025257588 A1 WO2025257588 A1 WO 2025257588A1
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Prior art keywords
tumor
cells
cancer
treatment
binding molecule
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Inventor
Erich Rajkovic
Holger DULAT
Daniela MORALES-ESPINOSA
Jens PAHL
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Affimed GmbH
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Affimed GmbH
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Publication of WO2025257588A1 publication Critical patent/WO2025257588A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • 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
    • 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
    • C07K16/283—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 Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • the present invention relates a cancer treatment method for immune checkpoint inhibitor resistant patients that uses a polyspecific binding molecule that binds to a CD16A surface antigen on an innate immune cell, and a surface antigen on a tumor cell. Treatment can also use a PD-1 or PD-L1 inhibitor on cells sensitized by the polyspecific binding molecule.
  • Immune checkpoint inhibitors have been used to treat many types of cancers, including non-small cell lung cancer, melanoma, renal cell carcinoma, head and neck cancer, and gastrointestinal cancer. ICIs function to block the inactivity of immune cells towards tumor cells. Normally, immune cells are controlled under homeostatic conditions using immune checkpoints which maintain a balance between pro-inflammatory and antiinflammatory signals. ICIs can promote anti-cancer immune responses, shifting the balance to proinflammatory, by binding to immune inhibitory receptors, such as CTLA-4, PD-1, and PD-L receptors on the surface of T-lymphocytes, and allowing activation of the T- lymphocytes towards the tumor. By blocking the immune inhibitory receptors, the ICIs overcome tumor-mediated immune inhibition, and facilitate a local inflammatory anti-tumor effect.
  • immune inhibitory receptors such as CTLA-4, PD-1, and PD-L receptors
  • ICIs target PD-1/PD-L1, which are immune-checkpoint molecules that inhibit the activation of antigen-presenting cells and T cells (priming phase) and the cytotoxic functions of T cells (effector phase).
  • Nivolumab (Bristol Myers Squibb), is a human IgG4 monoclonal antibody that blocks PD-1.
  • Ipilimumab is another immune- checkpoint monoclonal antibody that binds to CTLA-4, which is involved in the priming phase, has been approved for the treatment of NSCLC.
  • Other anti-PD-l/PD-Ll antibody monotherapy that is either currently approved and under development is shown in Table 1 of Mizuno T, Katsuya Y, Sato J, et al. Emerging PD-1/PD-L1 targeting immunotherapy in non- small cell lung cancer: Current status and future perspective in Japan, US, EU, and China. Frontiers in Oncology. 2022; 12:925938.
  • the present invention is based at least partly on the surprising finding that in clinical studies associated with the current disclosure, a combination therapy of a polyspecific binding molecule that, at least, binds CD16A on innate immune cells and a tumor antigen, e.g., EGFR or FOLR1, on tumor cells and a PD-L1 antibody (atezolizumab) can be used to effectively treat solid tumors, e.g. EGFR-positive and/or FOLR1 -positive solid tumors, in patients that have previously been demonstrated to be resistant to immune checkpoint inhibitor cancer treatment.
  • a combination therapy of a polyspecific binding molecule that, at least, binds CD16A on innate immune cells and a tumor antigen, e.g., EGFR or FOLR1, on tumor cells and a PD-L1 antibody e.g. EGFR-positive and/or FOLR1 -positive solid tumors
  • the polyspecific binding molecule Upon administration, the polyspecific binding molecule engages NK cells, and caused secretion of cytokines, and in turn induces changes in the tumor-microenvironment, which promoted re-activation of previously inactive T cells or engagement of further tumorspecific T cells.
  • administering the polyspecific binding molecule transformed a “cold tumor” (unresponsive) into a “hot tumor” (responsive), which is sensitized for ICI treatment again.
  • Administering the polyspecific binding molecule also caused T cells to migrate back into the tumor as evidenced by T cell activation, and a corresponding reduction in tumor mass was observed.
  • NK cell T cell
  • cytotoxicity associated functional genes such as Granzyme
  • PRs partial responses
  • CRs complete response
  • the polyspecific binding molecule also allowed the cancer cells to regain sensitivity to immune checkpoint inhibitor cancer treatment (e.g., PD-1 or PD-L1 antibody treatment).
  • the polyspecific binding molecule e.g., anti-CD16A/anti-EGFR
  • the combination therapy can be used to treat subjects that are not presently resistant, but that would be expected to develop resistance to checkpoint inhibitor treatment.
  • the treatment steps of the disclosure can also be used to treat tumors characterized by surface antigens other than EGFR in patients having previously been demonstrated to be resistant to immune checkpoint inhibitor cancer treatment.
  • the invention provides a method of treating a solid tumor in a subject, wherein the subject is, or is predicted to be, resistant to immune checkpoint inhibitor cancer treatment.
  • the method includes administering to the subject a polyspecific binding molecule that, at least, binds CD16A on innate immune cells and a tumor antigen on tumor cells, and also administering to the subject an immune checkpoint inhibitor, such as a PD-1 or PD-Ll inhibitor
  • the solid tumor is characterized by epidermal growth factor receptor (EGFR) overexpression on solid tumor cells, and the polyspecific binding molecule binds to EGFR.
  • EGFR epidermal growth factor receptor
  • the polyspecific binding molecule may not elicit EGFR downstream signalling and/or the EGFR-expressing solid tumors do not have driver or resistance mutation(s).
  • the solid tumor cells are characterized by folate receptor 1 (FOLR1) overexpression on their surface, and the poly specific binding molecule binds to FOLR1.
  • the polyspecific binding molecule may not elicit EGFR downstream signalling and/or the EGFR- expressing solid tumors do not have driver or resistance mutation(s).
  • the subject for which treatment is given can be a patient who has already received and has been found to be resistant to immune checkpoint inhibitor cancer treatment.
  • the subject can be one that was treated with ipilimumab, pembrolizumab, nivolumab, cemiplimab, dostarlimab, atezolizumab, durvalumab, or avelumab, and has been found to be resistant to one or more of these biologic drugs.
  • the subject has previously undergone treatment with a non-biologic, such as a platinum-based therapeutic treatment, and a immune checkpoint inhibitor.
  • the subject has an actionable driver mutation other than an EGFR mutation, and has undergone prior treatment for the mutation.
  • the actionable driver mutation other than an EGFR mutation is a targetable mutation in one or more of ERBB2 (HER2), ALK, ROS1, RET, NTRK, MET, BRAF, and KRAS genes.
  • the invention provides a pharmaceutical composition for treating cancer in a subject, the composition comprising a mixture of a polyspecific binding molecule that at least binds CD16A on innate immune cells and a tumor antigen on tumor cells, and a PD-L1 inhibitor, wherein the PD-L1 inhibitor is present in an amount that is greater than an amount of polyspecific binding molecule.
  • the invention provides a method of re-sensitizing a subject to immune checkpoint inhibitor cancer treatment.
  • the method includes administering to a subject who is, or is predicted to be, resistant to immune checkpoint inhibitor cancer treatment a polyspecific binding molecule that, at least, binds CD16A on innate immune cells and a tumor antigen on tumor cells.
  • the subject can be assessed for tumor regression, such as after each cycle of treatment. Treating can result in measurable shrinkage of tumor, as cytotoxic T cells to migrate into the tumor and cause a reduction in tumor mass. In patients receiving this therapy, partial responses (PRs) and complete response (CRs) can be seen, revealing that the disease had stabilized.
  • PRs partial responses
  • CRs complete response
  • Figures 1 Cytokines (IFNy (A), IL 10 (B), TNFa (C)) pre-dose Boxplot of percentage change from baseline in pre-dose measurements versus cycle /day by dose group. Low dose: 14 mg - 80 mg vs high dose: 160 mg - 720 mg), log scale. Dots: individual measurements, colored per dose cohort. Shown is the mean increase of pre-dose measured cytokines vs baseline. Baseline is pre-dose measurement prior to the first AFM24 dose (C1D1) and set to 100%.
  • C1D1 AFM24 dose
  • Figure 2 Immune cell populations in isolated PBMCs and relevant activation markers have been longitudinally analyzed by CyTOF. Cell populations have been identified by Boolean gating. Box plots showing A) the CD56dim NK cell frequency in CD45+ cells, B) the frequency of Ki-67+ CD56dim NK cells, C) the geometric mean of CD 16 on CD56dim NK cells, D) the CD8 T cell frequency in CD45+ cells, and E) the frequency of Ki-67+ CD8 T cells.
  • Dots are individual patient measurements where data from low dose (14 mg - 80 mg) cohorts are depicted as unfilled dots and data from high dose (160 mg - 720 mg) cohorts are shown as black dots.
  • Figure 3 EGFR staining of paired biopsies.
  • the baseline biopsy was taken at screening of the patient, while the second biopsy was taken after the 3rd dosing of AFM24 (C1D24).
  • the H score is calculated from a combination of staining intensity and staining area and ranges from 0 to 300.
  • Figure 4 Quantification of macrophages in paired biopsies from screening and at C1D24. Cohorts were grouped by colour. The red cross indicates the mean measurement.
  • FIG. 1 Box plot of all macrophages as stained with CD68
  • B Box plot of M2 macrophages as stained with CD 163.
  • Figure 5 Gene expression profiling and IHC of biopsies indicate an increase in cytotoxic cells within the tumor
  • Biopsies are variable and reflect only a snapshot of analyzed patient samples. An increase in CD3 positive cells in the tumor area could be detected.
  • Tumor biopsies were taken at screening and C1D24 of patients treated with AFM24 at doses higher than 160 mg and further analyzed by IHC and gene expression profiling using the Nanostring nCounter® PanCancer Immune Profiling Panel.
  • Figure 9 A Patient exhibiting a PR experienced a 50% overall shrinkage of their target lesions with AFM24 and Atezolizumab. Tumor Response by Investigator Assessment per RECIST vl.l and CT scan images of the RUL lesion of a patient exhibiting a confirmed PR on 480 mg AFM24 + Atezolizumab.
  • Figure 10 A Patient exhibiting a PR experienced a 35% overall shrinkage of their target lesions with AFM24 and Atezolizumab. Tumor Response by Investigator Assessment per RECIST vl.l. CT scan images of the LUL lesion (arrows in upper panel) and the left adrenal (arrows in lower panel) of a patient exhibiting a confirmed PR on 480 mg AFM24 + Atezolizumab.
  • Figure 14 A Partial Response was observed for this patient the present study. Patient was on SD until TA4 (-14.9% compared to baseline). Change in SOD compared to baseline: - 40%. Non-Target lesions were evaluated as: NTL1 : without changes; NTL2: slight decrease; NTL3 : decreased.
  • Figure 16 Antibody-induced release of IL-6, TNF-a, and IFN-y in human PBMC cultures in presence of EGFR-positive target cells after 4h.
  • Human PBMC were cultured in the presence or absence of EGFR-positive A-431 cells at an E:T ratio of 50: 1 with or without increasing concentrations of AFM24, scFv-IgAb_44 (anti-RSV/CD16A), scFv-IgAb_45 (anti- EGFR/RSV), cetuximab, or T cell (CD3/CD28) activator beads.
  • FIG. 17 Antibody-induced release of IL-6, TNF-a, and IFN-y in human PBMC cultures in presence of FOLR1 -positive target cells after 4h.
  • Human PBMC were cultured in the presence or absence of 100 pg/mL antibody (AFM32), IgAb_335 (human IgGl anti-FRa), scFv-IgAb_162 (FRa/RSV) or scFv-IgAb_444 (RSV/CD16A)) and in the presence or absence of FRa-positive OVCAR-3 cells.
  • AFM32 antibody to human PBMC
  • IgAb_335 human IgGl anti-FRa
  • scFv-IgAb_162 FRa/RSV
  • scFv-IgAb_444 RSV/CD16A
  • FIG. 18 In vitro ADCP of DK-MG cells and HCT-116 cells mediated by AFM24 via human macrophages.
  • In vitro generated human macrophages were co-incubated with CMFDA-labeled DK-MG cells (A) or HCT-116 cells (B) at an E:T ratio of 5: 1 in the presence or absence of lOpg/mL or O.lpg/mL of AFM24 or control antibodies.
  • ADCP was assessed by flow cytometry by quantifying CD1 lb+CMFDA+ cells, and normalization to the w/o antibody control. Values are means +/- standard deviation (SD).
  • FIG. 19 In vitro ADCP of tumor (HeLa and HCC-78) and NK cells mediated by human macrophages.
  • In vitro generated macrophages cells were co-cultured with CMFDA- labeled HeLa (A), HCC-78 (B), or NK cells (C) at an E:T ratio of 5: 1 in the presence of increasing concentrations of AFM32, scFv-IgAb_444 (RSV/CD16A), scFv-IgAb_162 (FRa/RSV), IgAb_335 (anti-FRa IgGl), and IgAb_346 (Fc-silenced IgGl anti-FRa).
  • ADCP was assessed by flow cytometry after 4 h incubation. The figure shows the results of one of three replicate experiments.
  • FIG. 20 Adoptive transfer of NK cells in combination with AFM24 induces an AFM24 dose-dependent tumor growth regression in vivo.
  • the administration schema for the in vivo model is depicted in (A).
  • FIG. 21 In vivo Dose-Titration Study of AFM32 in B-hCD16A mice (CB-17 SCID) bearing SKOV3 Red FLuc Subcutaneous Xenograft Tumors.
  • A Schematic Diagram of Study Design
  • B Tumor growth curves of treatment groups in SKOV3 Red FLuc tumorbearing B-hCD16A (CB-17 SCID) mice. Tumor volume change from the day of inoculation. Day 18 baseline. The data are presented as the mean ⁇ SEM.
  • SKOV3 Red FLuc cells (5 xlO 6 cells per mouse) were subcutaneously injected on Day 0.
  • the current disclosure describes approaches for treating cancer in a subject, wherein the subject is, or is predicted to be, resistant to immune checkpoint inhibitor cancer treatment.
  • the current disclosure also describes approaches for re-sensitizing a subject to immune checkpoint inhibitor cancer treatment.
  • _a subject who is, or is predicted to be, resistant to immune checkpoint inhibitor cancer treatment is administered a polyspecific binding molecule that, at least, binds CD16A on innate immune cells and a tumor antigen on tumor cells.
  • the method includes administering to the subject a polyspecific binding molecule that, at least, binds CD16A on innate immune cells and a tumor antigen on tumor cells.
  • the treatment with the polyspecific binding molecule re-activates the adaptive and/or innate immune system through changes in the tumor microenvironment, which can be associated with the re-activation of tumor-specific T cells, thereby re-sensitizes the tumor to cancer treatment.
  • the polyspecific binding molecule can render the tumor in the subject sensitive again to treatment with an immune checkpoint inhibitor molecule, such as PD-L1 antibody treatment.
  • the polyspecific binding molecule engages NK cells, and causes secretion of cytokines thereby inducing changes in the tumor-microenvironment and sensitizing the tumor microenvironment to treatment with an ICI inhibitor.
  • increases in one or more or all of INFy, TNFa, IL- 10, IL-6, and/or IL- 15 can be observed in patients having at least one does of the poly specific binding molecule.
  • the first dose of the poly specific binding molecule may result in a substantial increase in one of more of these cytokines, and one or more subsequent dose(s) following the initial dose may not promote an increase in the one of more of these cytokines in the same amount as the initial dose.
  • the response curve of cytokine production can flatten after the initial dose.
  • upregulation of NK cell specific genes including CD69 and Ki-67 following administration of the initial dose of polyspecific binding molecule CD69 is an early activation marker, and increases in its expression after NK cell stimulation is considered a bona fide marker of NK cell activation
  • the polyspecific binding molecule promotes re-activation of previously inactive T cells or engagement of further tumor-specific T cells.
  • treatment with the polyspecific binding molecule leads to an indirect activation of T cells. T cell activation is more pronounced at higher doses polyspecific binding molecule and occurs later during treatment relative to NK cell activation. An increase in T cell and cytotoxicity associated functional genes, such as Granzyme, is observed.
  • subject that is treated has tumor that is characterized by epidermal growth factor receptor (EGFR) overexpression on the surface of the solid tumor cells.
  • EGFR epidermal growth factor receptor
  • the polyspecific binding molecule binds to EGFR on those tumor cells and also to CD16A on Natural Killer (NK) cells.
  • Subjects of the disclosure that can be treated using methods of the disclosure include those that have undergone “previous treatment” using at least an immune checkpoint inhibitor (ICI).
  • Previous treatment refers herein to tumor treatment that occurs prior to treatment of the disclosure using the polyspecific binding molecule.
  • the subject may have been diagnosed with one or more of cancer(s), such as non-small cell lung cancer, melanoma, renal cell carcinoma, head and neck cancer, or gastrointestinal cancer, colorectal cancer; cutaneous squamous cell carcinoma, esophageal squamous cell carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, head and neck squamous cell carcinoma, primary mediastinal B cell lymphoma, extensive- stage small cell lung cancer; TMB, tumor mutation burden-high; TNBC, triple-negative breast cancer.
  • cancer(s) such as non-small cell lung cancer, melanoma, renal cell carcinoma, head and neck cancer, or gastrointestinal cancer, colorectal cancer
  • cutaneous squamous cell carcinoma esophageal squamous cell carcinoma, hepatocellular carcinoma
  • Hodgkin lymphoma head and neck squamous cell carcinoma
  • TMB tumor mutation burden-high
  • TNBC triple-negative breast cancer.
  • the subject may not show satisfactory results, such as a partial unsatisfactory response to the ICI treatment, or no observable response to the ICI treatment.
  • the subject may also be one that shows satisfactory results at the onset of treatment, but that becomes less responsive to the treatment after a period of time.
  • the previous ICI treatment can use an ICI that binds to immune inhibitory receptors, such as CTLA-4, PD-1, and PD-L receptors.
  • the subject may have had a previous ICI treatment with an immune checkpoint inhibitor such as ipilimumab, pembrolizumab, nivolumab, cemiplimab, dostarlimab, atezolizumab, durvalumab, and avelumab.
  • an immune checkpoint inhibitor such as ipilimumab, pembrolizumab, nivolumab, cemiplimab, dostarlimab, atezolizumab, durvalumab, and avelumab.
  • the subject receiving previous treatment may have also been treated with one or more traditional chemotherapeutic drugs.
  • the previous treatment may have used one or more platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin. Patients previously treated with platinum-based drugs may have had their treatment discontinued due to systemic toxicity and drug resistance.
  • the subject receiving previous treatment may have also been treated with one or members of the taxane class of drugs such as paclitaxel (taxol), docetaxel (taxotere), and cabazitaxel.
  • taxane class of drugs such as paclitaxel (taxol), docetaxel (taxotere), and cabazitaxel.
  • These types of drugs_disrupt microtubule function which are critical to cell division, and inhibit the process of cell division by preventing as depolymerization.
  • These drugs are also widely used to treat a variety of solid tumors.
  • Patients previously treated with taxane class of drugs may have had their treatment discontinued due to toxicities, such as neutropenia, neuropathy, hypersensitivity, and alopecia.
  • taxanes are administered by lenghty IV infusions, which are difficult for many patients.
  • the subject receiving previous treatment may have also been treated with one or more antimetabolite drugs such as methotrexate, pemetrexed, raltitrexed and pralatrexate.
  • Patients previously treated with taxane class of drugs may have had their treatment discontinued due to side effects such as low blood cell count, fatigue, nausea, and gastrointestinal problems.
  • Patients having previous treatments may have undergone treatments with different drugs, or drug combinations, at different times. For example, some patients may have undergone two or more therapies using different drugs of drug combinations, wherein the therapies were discontinued as not providing desired results.
  • the patient to be treated with the polyspecific binding molecule according to the method of the disclosure was not previously treated with an ICI, but is found to be “primarily resistant” to treatment with an ICI. That is, a subject who is “primarily resistant” to treatment with an ICI does not have acquired resistance otherwise promoted by ICI treatment, but has physiological factors that are predictive to ICI resistance. See, for example, Nagasaki, J., et al. (Cancer Science. 2022;113:3303-3312).
  • Primary resistance mechanisms to ICI can be found, generally, in (a) resistance related to antigen recognition during T-cell activation when cancer antigen is presented by antigen presenting cells; (2) resistance related to antigen recognition during T-cell recognition of cancer antigen presented in the context of MHC molecules of cancer cells; and (3) resistance related to effector function of T cells.
  • TME tumor microenvironment
  • the presence of inhibitory immune checkpoint molecules LAG-3, TIM-3, and TIGIT, on T cells, which are not fully reactivated by blocking only PD-1, can be determined to predict primary resistance. Also, one may also determine if there are abnormalities in the IFN-y signaling pathway to determine primary resistance to ICIs. For example, it has been found that JAK1/2 loss-of-function mutations are a genetic mechanism of lack of reactive PD-L1 expression and response to interferon gamma, leading to primary resistance to PD-1 blockade therapy. See, for example, Shin, D.S., et al. Primary resistance to PD-1 blockade mediated by JAK1/2 mutations. Cancer Discov. 2017;7: 188-201.
  • the subject has an actionable driver mutation other than an EGFR mutation, and has undergone prior treatment for the mutation.
  • the actionable driver mutation other than an EGFR mutation is a targetable mutation in one or more of ERBB2 (HER2), ALK, ROS1, RET, NTRK, MET, BRAF, and KRAS genes.
  • An actionable mutation is a DNA alteration that can affect a patients’ response to treatments. The most relevant ones from a clinical standpoint can be targeted with specific drugs or therapies which are known as targetable mutations.
  • Targetable mutations in various oncodriver genes have been associated with non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • Patients with targetable mutations do not all respond equally to targeted therapies, and resistance can eventually develop. See, for example, Le, X., et al. New Actions on Actionable Mutations in Lung Cancers. Cancers (Basel). 15:2917, 2023.
  • the “Polyspecific Binding Molecule” (which can also be referred to as the “PBM”) is a molecule that includes, in the least, two binding domains, one binding domain (e.g., a first binding domain) for binding CD16A on the surface of the T cell, and another binding domain (e.g., a second binding domain), for binding an antigen on the surface of the tumor cell.
  • the PBM can optionally include third, fourth, fifth, etc., binding domains.
  • binding domain characterizes in connection with the present invention a domain which is capable of specifically binding to / interacting with / recognizing a given target epitope or a given target site on the target molecules (antigens), i.e.
  • CD16A on the surface of an immune effector cell, and a tumor cell surface antigen, respectively.
  • the structure and/or function of the first binding domain (recognizing CD16A), and also the structure and/or function of the second binding domain (recognizing the target cell surface antigen, e.g. EGFR or FOLR1), is/are preferably based on the structure and/or function of an antibody, e.g. of a full-length or whole immunoglobulin molecule and/or is/are drawn from the variable heavy chain (VH) and/or variable light chain (VL) domains of an antibody or fragment thereof.
  • VH variable heavy chain
  • VL variable light chain
  • binding domain preferentially binds or recognizes the target even when the binding partner is present in a mixture of other molecules or other structures.
  • the binding may be mediated by covalent or non-covalent interactions or a combination of both.
  • “simultaneous binding to a target cell and an immune effector cell” comprises the physical interaction between the binding domains and their targets on the cells, but preferably also includes the induction of an action mediated by the simultaneous binding of the two cells. Such an action may be an immune effector function of the immune effector cell, such as a cytotoxic effect.
  • antibody construct refers to a molecule in which the structure and/or function is/are based on the structure and/or function of an antibody, e.g., of a full-length or whole immunoglobulin molecule and/or is/are drawn from the variable heavy chain (VH) and/or variable light chain (VL) domains of an antibody or fragment thereof.
  • VH variable heavy chain
  • VL variable light chain
  • An antibody construct is hence capable of binding to its specific target or antigen.
  • the binding region of an antibody construct comprises the minimum structural requirements of an antibody which allow for the target binding. For the first binding domain to CD16A, this minimum requirement is defined by the presence of a VL region comprising the three light chain CDRs (i.e.
  • this minimum requirement may, e.g., be defined by the presence of at least the three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and/or the three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region), preferably of all six CDRs.
  • An alternative approach to define the minimal structure requirements of an antibody is the definition of the epitope of the antibody within the structure of the specific target, respectively, the protein domain of the target protein composing the epitope region (epitope cluster) or by reference to a specific antibody competing with the epitope of the defined antibody.
  • the antibodies on which the constructs defined in the context of the invention are based include for example monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies.
  • the first binding domain of an antibody construct comprises the designated groups of CDRs.
  • Those CDRs can be comprised in the framework of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the second binding domain comprises defined groups of CDRs.
  • those CDRs are comprised in the framework of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not have to comprise both.
  • Fd fragments for example, have two VH regions and often retain some antigen-binding function of the intact antigen-binding region.
  • Examples for the format of antibody fragments, antibody variants or binding domains include (1) a Fab fragment, a monovalent fragment having the VL, VH, CL and CHI domains; (2) a F(ab')2fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge domain; (3) an Fd fragment having the two VH and CHI domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which has a VH domain; (6) an isolated complementarity determining region (CDR), and (7) a single chain Fv (scFv), the latter being preferred (for example, derived from an scFv-library).
  • a Fab fragment a monovalent fragment having the VL, VH, CL and CHI domains
  • F(ab')2fragment a bivalent fragment having two Fab
  • Examples for embodiments of antibody constructs according to the invention are e.g. described in WO00/006605, W02005/040220, W02008/119567, W02010/037838, WO2013/026837, WO2013/026833, US2014/0308285, US2014/0302037, WO2014/144722, W02014/151910, and WO20 15/048272.
  • An antibody construct that can be used in methods of the disclosure can comprise a fragment of a full-length antibody, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or "r IgG" ("half antibody").
  • Antibody constructs can also comprise modified fragments of antibodies, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv- zipper, scFab, Fab2, Fabs, diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "multibodies” such as triabodies or tetrabodies, and single domain antibodies such as nanobodies or single variable domain antibodies comprising merely one variable domain, which might be VHH, VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains.
  • antibody variants such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv- zipper, scFab, Fab2, Fabs, diabodies, single chain diabodies, tandem di
  • single-chain Fv single polypeptide chain antibody fragments that comprise the variable regions from both the heavy and light chains, but lack the constant regions.
  • a single-chain antibody further comprises a polypeptide linker between the VH and VL domains which enables it to form the desired structure which would allow for antigen binding.
  • linkers for this purpose include glycine serine linkers, which preferably comprises from about 15 to about 30 amino acids.
  • Preferred glycine serine linkers may have one or more repeats of GGS, GGGS, or GGGGS.
  • Such linker preferably comprises 5, 6, 7, 8, 9 and/or 10 repeats of GGS, preferably (GGS)e (which are preferably used for scFvs having the arrangement VH-VL), or preferably (GGS)? (which are preferably used for scFvs having the arrangement VL-VH).
  • GGS preferably (GGS)e (which are preferably used for scFvs having the arrangement VH-VL), or preferably (GGS)? (which are preferably used for scFvs having the arrangement VL-VH).
  • GGS preferably (GGS)e (which are preferably used for scFvs having the arrangement VH-VL), or preferably (GGS)? (which are preferably used for scFvs having the arrangement VL-VH).
  • GGS preferably (GGS)e (which are preferably used for scFvs having the arrangement VH-VL), or preferably (GGS)? (which are preferably used for
  • single-chain antibodies can also be human, and/or humanized and/or synthetic.
  • the term “bi-scFv” or “ta-scFv” (tandem scFv) as used herein refers to two scFv that are fused together.
  • Such a bi-scFv or ta- scFv may comprise a linker between the two scFv moieties.
  • the arrangement of the VH and VL domains on the polypeptide chain within each of the scFv may be in any order.
  • the “bi-scFv” of “ta-scFv” can be arranged in the order VH(1)-VL(1)-VH(2)- VL(2), VL(1)-VH(1)-VH(2)-VL(2), VH(1)-VL(1)-VL(2)-VH(2), or VL(1)-VH(1)-VL(2)- VH(2), where (1) and (2) stand for the first and second scFv, respectively.
  • double Fab refers to two Fab fragments that are fused together, which are preferably staggered.
  • a first chain of a first Fab is N-terminally fused to a first chain of a second Fab, or a second chain of a first Fab is N-terminally fused to a second chain of a second Fab, or both, the first chain of a first Fab and the second chain of a first Fab are fused to first and second chains of a second Fab, respectively.
  • a linker may be present between the fused chains of the first and second Fab.
  • the first and second chains of the first and second Fab can be individually selected from a light chain-derived chain of a Fab (VL-CL), a heavy chain derived chain of a Fab (VH-CH1), as long as each Fab contains a VH, a VL, a CHI, and a CL.
  • VL-CL light chain-derived chain of a Fab
  • VH-CH1 heavy chain derived chain of a Fab
  • the light chain-derived chain of the first Fab can be fused to the light chain derived-chain of the second Fab.
  • the heavy chain- derived chain of the first Fab can be fused to the heavy chain derived-chain of the second Fab.
  • the heavy chain-derived chain of the first Fab can be fused to the light chain derived-chain of the second Fab.
  • both chains of the two Fabs are fused together.
  • the light chain-derived chain of the first Fab can be fused to the light chain derived-chain of the second Fab while the heavy chain-derived chain of the first Fab can be fused to the heavy chain derived-chain of the second Fab.
  • the light chain-derived chain of the first Fab can be fused to the heavy chain derived-chain of the second Fab while the heavy chain-derived chain of the first Fab can be fused to the light chain derived-chain of the second Fab.
  • the binding domains and the variable domains (VH/VL) of the antibody construct of the disclosure may or may not comprise peptide linkers (spacer peptides).
  • a "peptide linker" is an amino acid sequence by which the amino acid sequences of one (variable and/or binding) domain and another (variable and/or binding) domain of the antibody construct defined herein are linked with each other.
  • the peptide linkers can also be used to fuse one domain to another domain of the antibody construct defined herein. In such cases, the peptide linker may also be referred to as a “connector”.
  • Such a connector is preferably a short linker, which preferably has a length of about 10 nm or less, preferably about 9 nm or less, preferably about 8 nm or less, preferably about 7 nm or less, preferably about 6 nm or less, preferably about 5nm or less, preferably about 4 nm or less, or even less.
  • the length of the linker is preferably determined as described by Rossmalen, et al., (Biochemistry 56:6565-6574, 2017), which also describes suitable linkers that are well known to the skilled person.
  • An example for a connector is a glycine serine linker or a serine linker, which preferably comprise no more than about 75 amino acids, preferably not more than about 50 amino acids.
  • a fusion of two Fab chains may optionally comprise a linker.
  • Suitable and preferred linkers comprise the upper hinge sequence (EPKSCDKTHT) or glycine serine linkers with about up to 20 amino acids, preferably up to 10 amino acids, or most preferably 10 amino acids, e.g. two repeats of GGGGS.
  • Glycine serine linkers comprised in a double Fab may have one or more repeats of GGS, GGGS, or GGGGS, such as one, two, three, or four repeats.
  • a “diabody” or “Db” refers to an antibody construct comprising two binding domains, which may be constructed using heavy and light chains disclosed herein, as well as by using individual CDR regions disclosed herein.
  • a diabody comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.
  • linkers for this purpose include glycine serine linkers with about up to 12 amino acids, preferably up to about 10 amino acids.
  • Preferred glycine serine linkers may have one or more repeats of GGS, GGGS, or GGGGS.
  • a preferred linker is (GGS)2.
  • a diabody can be formed by two separate polypeptide chains, each comprising a VH and a VL.
  • all four variable domains can be comprised in one single polypeptide chain comprising two VH and two VL domains.
  • the diabody can also be termed “single chain diabody” or “scDb”.
  • a scDb comprises the two chains of a non-single chain diabody that are fused together, preferably via a linker.
  • a preferred linker for this purpose is a glycine serine linker, which preferably comprises from about 15 to about 30 amino acids.
  • Preferred glycine serine linkers may have one or more repeats of GGS, GGGS, or GGGGS.
  • Such linker preferably comprises 5, 6, 7, 8, 9, and/or 10 repeats of GGS, preferably (GGS)e, or preferably (GGS)?.
  • the variable domains of a scDb can be arranged (from N to C terminus) in a VL-VH-VL-VH or VH-VL-VH-VL order.
  • the spatial arrangement of the four domains in the tertiary/quaternary structure can be in a VL-VH-VL-VH or VH-VL-VH-VL order.
  • diabody does not exclude the fusion of further binding domains to the diabody.
  • antibody construct includes monovalent, bivalent and polyvalent / multivalent constructs, i.e. monovalent, bivalent, trivalent, or even higher valency for first and second target bound by the first and second binding domain, wherein the antibody construct is necessarily bispecific as described elsewhere herein, i.e. comprises specificities for two different antigens or targets.
  • the term “valent” denotes the presence of a determined number of antigen-binding domains in the antigen-binding protein. A natural IgG has two antigen-binding domains and is bivalent.
  • the bispecific antibody constructs of the present invention may comprise one, two or more first binding domains (A) against CD16A and one, two or more second binding domains (B) against a second target on the surface of a tumor cell.
  • antibody construct includes molecules consisting of only one polypeptide chain as well as molecules consisting of more than one polypeptide chain, which chains can be either identical (homodimers, homotrimers or homo oligomers) or different (heterodimer, heterotrimer or heterooligomer).
  • bispecific refers to an antibody construct which is "essentially bispecific", i.e., comprise specificities for two different antigens or targets, but no further specificity against a third or further antigen or target.
  • a bispecific antibody construct that can be used in methods of the disclosure has a (first) binding domain that binds to CD16A and a (second) binding domain that binds to a tumor antigen.
  • CD16A refers to the activating receptor CD16A, also known as FcyRIIIA, expressed on the cell surface of NK cells.
  • CD16A is an activating receptor triggering the cytotoxic activity of NK cells.
  • the amino acid sequence of human CD16A is given in UniProt entry P08637 (version 212 of 12 August 2020).
  • the affinity of antibodies for CD16A directly correlates with their ability to trigger NK cell activation, thus higher affinity towards CD16A reduces the antibody dose required for activation.
  • the antigen-binding site of the antigen-binding protein binds to CD16A, but preferably not to CD16B.
  • an antigen-binding site comprising heavy (VH) and light (VL) chain variable domains binding to CD 16 A, but not binding to CD16B, may be provided by an antigen-binding site which specifically binds to an epitope of CD16A which comprises amino acid residues of the C- terminal sequence SFFPPGYQ (positions 201-208 of CD16A) and/or residues G147 and/or Y158 of CD16A which are not present in CD16B.
  • CD16B refers to receptor CD16B, also known as FcyRIIIB, expressed on neutrophils and eosinophils.
  • the receptor is glycosylphosphatidyl inositol (GPI) anchored and is understood to not trigger any kind of cytotoxic activity of CD16B positives immune cells.
  • GPI glycosylphosphatidyl inositol
  • a tumor cell or “target cell” describes a cell or a group of cells, which is/are the target of the mode of action applied by the antibody construct of the disclosure. Tumor cells are eliminated or inhibited by engaging these cells with the effector cell via the antibody construct of the invention.
  • CD16A shedding refers to the down-modulation / down-regulation/ degradation of FcyRIIIA expressed on the cell surface of immune effector cells such as NK cells after binding and activation of immune effector cells by a CD16A binding domain, e.g. an antibody.
  • CD16A shedding is typically mediated by A disintegrin and metalloproteinase (ADAMI 7), or membrane type 6 matrix metalloproteinase (MMP25) and describes a proteolytic process that regulates the cell surface density of said surface molecules on immune effector cells.
  • ADAMI 7 A disintegrin and metalloproteinase
  • MMP25 membrane type 6 matrix metalloproteinase
  • CD16A shedding is known as activation-induced down-regulation as described e.g.
  • tumor cell surface antigen refers to an antigenic structure expressed by a tumor cell and which is present at the tumor cell surface such that it is accessible for an antibody construct as described herein. It may be a protein, preferably the extracellular portion of a protein, a peptide that is presented on the cell surface in an MHC context (including HLA-A2, HLA-A11, HLA-A24, HLA-B44, HLA-C4) or a carbohydrate structure, preferably a carbohydrate structure of a protein, such as a glycoprotein.
  • the antigen can be a tumor associated or tumor restricted antigen.
  • Target tumor cell surface antigens include, but are not limited to, EGFR, EGFRvIII, HER2, HER3, HER4, FOLR1, and GD2 as defined elsewhere herein.
  • CD16A used in the context of the disclosure is not a target tumor cell surface antigen of the present invention.
  • Bispecific antibody constructs can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315- 321 (1990)
  • the antibody constructs that can be used in methods of the disclosure are preferably "in vitro generated antibody constructs".
  • This term refers to an antibody construct according to the above definition where all or part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection, e.g., an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen.
  • a non-immune cell selection e.g., an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen.
  • a "recombinant antibody” is an antibody made through the use of recombinant DNA technology or genetic engineering.
  • mAb monoclonal antibody
  • monoclonal antibody construct refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic side or determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (or epitopes).
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, hence uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies and antibody constructs of the present disclosure specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 : 6851 -6855 (1984)).
  • chimeric antibodies immunoglobulins
  • Chimeric antibodies of interest herein include "primitized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences.
  • a non-human primate e.g., Old World Monkey, Ape etc.
  • human constant region sequences e.g., human constant region sequences.
  • a variety of approaches for making chimeric antibodies have been described. See e.g., Morrison et al., Proc. Natl. Acad. Sci U.S.A. 81 :6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.
  • An antibody, antibody construct, antibody fragment or antibody variant may also be modified by specific deletion of human T cell epitopes (a method called "deimmunization") by the methods disclosed for example in WO 98/52976 or WO 00/34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC class II; these peptides represent potential T cell epitopes (as defined in WO 98/52976 and WO 00/34317).
  • peptide threading For detection of potential T cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes.
  • Potential T cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable domains, or preferably, by single amino acid substitutions. Typically, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used.
  • Humanized antibodies are antibodies or immunoglobulins of mostly human sequences, which contain (a) minimal sequence(s) derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non- human (e.g., rodent) species (donor antibody) such as mouse, rat, hamster or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • "humanized antibodies” as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 : 522-525 (1986);
  • Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains.
  • Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229: 1202-1207; by Oi et al. (1986) BioTechniques 4:214; and by US 5,585,089; US 5,693,761; US 5,693,762; US 5,859,205; and US 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain.
  • Such nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources.
  • the recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.
  • Humanized antibodies may also be produced using transgenic animals such as mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539).
  • All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.
  • a humanized antibody can be optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and/or back mutations.
  • Such altered immunoglobulin molecules can be made by any of several techniques known in the art, (e.g., Teng, et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3- 16, 1982, and EP 239 400).
  • human antibody includes antibodies, antibody constructs and binding domains having antibody regions such as variable and constant regions or domains which correspond substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) (loc. cit.).
  • the human antibodies, antibody constructs or binding domains as defined in the context of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or side-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, in CDR3.
  • the human antibodies, antibody constructs or binding domains can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence.
  • a "fully human antibody” does not include amino acid residues not encoded by human germline immunoglobulin sequences.
  • the antibody constructs defined herein are "isolated" or "substantially pure” antibody constructs.
  • Isolated or substantially pure when used to describe the antibody constructs disclosed herein, means an antibody construct that has been identified, separated and/or recovered from a component of its production environment.
  • the antibody construct is free or substantially free of association with all other components from its production environment.
  • Contaminant components of its production environment such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the antibody constructs may e.g. constitute at least about 5%, or at least about 50% by weight of the total protein in a given sample.
  • the isolated protein may constitute from 5% to 99.9% by weight of the total protein content, depending on the circumstances.
  • the polypeptide may be made at a significantly higher concentration through the use of an inducible promoter or high expression promoter, such that it is made at increased concentration levels.
  • the definition includes the production of an antibody construct in a wide variety of organisms and/or host cells that are known in the art.
  • the antibody construct will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • an isolated antibody construct will be prepared by at least one purification step.
  • the antibody construct which can be used in methods of the disclosure can also include additional domains, which are e.g. helpful in the isolation of the molecule or relate to an adapted pharmacokinetic profile of the molecule.
  • Domains helpful for the isolation of an antibody construct may be selected from peptide motives or secondarily introduced moieties, which can be captured in an isolation method, e.g. an isolation column.
  • additional domains comprise peptide motives known as Myc-tag, HAT- tag, HA-tag, TAP -tag, GST-tag, chitin binding domain (CBD-tag), maltose binding protein (MBP-tag), Flag-tag, Strep-tag and variants thereof (e.g.
  • All herein disclosed antibody constructs characterized by the identified CDRs may comprise a His-tag domain, which is generally known as a repeat of consecutive His residues in the amino acid sequence of a molecule, preferably of five, and more preferably of six His residues (hexahistidine).
  • the His-tag may be located e.g. at the N- or C-terminus of the antibody construct, preferably it is located at the C-terminus.
  • a hexa-histidine tag is linked via peptide bond to the C-terminus of the antibody construct according to the invention.
  • sequence identity and/or similarity is determined by using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl.
  • Acid Res. 12:387-395 preferably using the default settings, or by inspection.
  • percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360; the method is similar to that described by Higgins and Sharp, 1989, CAB IOS 5: 151 -153.
  • Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • Another example of a useful algorithm is the BLAST algorithm, described in: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389- 3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787.
  • a particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions, charges gap lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to about 22 bits.
  • amino acid homology, similarity, or identity between individual variant CDRs or VH / VL sequences are at least 60% to the sequences depicted herein, and more typically with preferably increasing homologies or identities of at least 65% or 70%, more preferably at least 75% or 80%, even more preferably at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100%.
  • percent (%) nucleic acid sequence identity with respect to the nucleic acid sequence of the binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the antibody construct.
  • a specific method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • nucleic acid sequence homology, similarity, or identity between the nucleotide sequences encoding individual variant CDRs or VH / VL sequences and the nucleotide sequences depicted herein are at least 60%, and more typically with preferably increasing homologies or identities of at least 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and almost 100%.
  • a "variant CDR” or a “variant VH / VL region” is one with the specified homology, similarity, or identity to the parent CDR / VH / VL defined in the context of the invention, and shares biological function, including, but not limited to, at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or activity of the parent CDR or VH / VL.
  • EGFR refers to the epidermal growth factor receptor (EGFR; ErbB-1;
  • the EGFR antigen-binding site recognizes an epitope in the extracellular domain of the EGFR. In certain embodiments the antigen-binding site specifically binds to human and cynomolgus EGFR.
  • the epidermal growth factor receptor (EGFR) is a member of the HER family of receptor tyrosine kinases and consists of four members: EGFR (ErbBl/HERl), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Stimulation of the receptor through ligand binding (e.g.
  • EGF EGF, TGFa, HB-EGF, neuregulins, betacellulin, amphiregulin
  • EGFR can be expressed by a number of different tumor types including lung (particularly non-small cell cancer NSCLC), colorectal (particularly CRC), liver (particularly hepatocellular carcinoma HCC), brain (particularly glioblastoma GBM), kidney/renal (particularly ccRCC), ovarian, breast (particularly TNBC), squamous cell carcinoma (particularly squamous cervical cancer), bladder, head and neck (particularly squamous cell carcinoma of head and neck SCCHN), gastric cancer, esophagus, sarcoma, mesothelioma, and adenocarcinoma.
  • FOLR1 also known as “Folate Receptor 1”, “Folate Receptor Alpha”, “Folate Binding Protein” (FBP” or “Ovarian Cancer-Associated Antigen” is a receptor with high affinity for folic acid and for several reduced folic acid derivatives and mediates delivery of 5-methyltetrahydrofolate to the interior of cells (Henderson, Annu Rev Nutr. 1990; 10:319-35).
  • the human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot.
  • the amino acid sequence of human FOLR1 can be found as UniProt/Swiss-Prot Accession No.
  • Pl 5328 (version 204 of 29 May 2024) and the nucleotide sequence encoding of the human FOLR1 can be found at Accession No. BT007158.1 (version BT007158.1 of 13 May 2003).
  • FOLR1 can be overexpressed by a number of epithelial-derived tumors including ovarian, breast (particularly TNBC), , lung, colorectal (CRC), kidney/renal (particularly ccRCC), pancreatic (particularly PDAC), endometrial and brain (Scaranti et al., Nat Rev Clin Oncol. 2020, 17(6): 349-359).
  • F0LR1 includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full length wild-type F0LR1.
  • GD2 refers to a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans (Nazha et al., Front Oncol, 2020, 10: 1000).
  • Poly specific binding molecule having a first binding domain against CD 16 A, and a second binding domain against EGFR, EGFRvIII, HER2, HER3, HER4, FOLR1, or GD2, can be prepared and used in the method of the disclosure.
  • CDRs from antibodies against EGFR, EGFRvIII, HER2, HER3, HER4, FOLR1, and GD2, are known in the art and can be incorporated into a poly specific binding molecule along with CDRs against CD 16 A.
  • Antibodies against EGFR are e.g. described in W09520045, WO9525167, and W002066058.
  • Antibodies against EGFRvIII are e.g. described in WO2017125831.
  • Antibodies against HER2 are e.g. described in US2011189168, W00105425, and US2002076695.
  • Antibodies against GD2 are e.g. described in W08600909, W08802006, and US5977316.
  • Antibodies against FOLR1 are well known in the art and are e.g. described in US9522196B2 and W02014104270.
  • anti-human FOLR1 antibodies are farletuzumab (INN number 9067 - sequences have also been disclosed under drugbank.ca/drugs/DB05595/polypeptide_sequences.fasta and the IMTG database (imtg.org under the INN number 9067)) and mirvetuximab (INN number 10187 - sequence has been disclosed in the IMTG database (imtg.org under the INN number 10187)).
  • the method of the disclosure uses a poly specific binding molecule having a first binding domain against CD 16 A, and a second binding domain against EGFR, EGFRvIII, HER2, HER3, HER4, FOLR1, or GD2, can be prepared and used in the method of the disclosure.
  • the method of the disclosure uses a bispecific binding molecule having a first binding domain against CD 16 A, and a second binding domain against EGFR, EGFRvIII, HER2, HER3, HER4, FOLR1, and GD2, as taught in WO2023/078968, the disclosure of which is incorporated herein by reference.
  • the bispecific binding molecule of WO2023/078968 include high-affinity anti-CD16A first binding domain and a second binding domain for a tumor antigen, which can efficiently activate and redirect immune effector cells for ADCC, thereby avoiding CD16A shedding and immediate inactivation of the engaged effector cells.
  • SEQ ID NOs: 1-179 of WO2023/078968 are incorporated herein by reference, and in particular see VH, VL, HCDR1-3, LCDR1-3 scDB, and scFv sequences of SEQ ID NOs 1-20 and 124-153 for CD16A and EGFR.
  • the method of the disclosure uses a trispecific binding molecule having a first binding domain against CD 16 A, and a second binding domain against CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, SLAMF7, 0X40, CD47/SIRPa, CD89, CD96, CD 137, CD 160, TIGIT, nectin-4, PD-1, PD-L1, LAG-3, CTLA- 4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, and CD3, and a third binding domain against EGFR, EGFRvIII, HER2, HER3, HER4, FOLR1, and GD2, as taught in W02022/074206, the disclosure of which is incorporated herein by reference.
  • Exemplary constructs include anti-EGFR/NKp46/CD16 and anti-EGFR/CD16A/NKG2D constructs.
  • SEQ ID NOs: 1-502 of W02022/074206 are incorporated herein by reference, and in particular see VH, VL, HCDR1-3, and LCDR1-3 sequences of SEQ ID NOs 1-12, 15-49, and 56-79, for CD16A, EGFR, NKp46, and NKG2D.
  • the method of the disclosure uses a poly specific binding molecule having at least four first binding domains, with a first binding domain that binds to an immune-regulatory antigen on the surface of a natural killer cell or a macrophage (e.g., CD16A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), 0X40, CD47, SIRPa, CD89, CD96, CD137, CD160, TIGIT, nectin-4, PD-1, PD-L1, LAG-3, CTLA-4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1, and CD3) and a second binding domain binding to a second target that is an antigen on the surface of a tumor cells (EGFR, EGFRvIII, HER2, HER3,
  • a tumor cells e
  • W02023/007023 teaches bispecific antibodies having one or two binding domains for EGFR and four binding domains for CD16A have a surprisingly increased potency and efficacy against Daudi cells.
  • SEQ ID NOs: 1-194 of W02023/007023 are incorporated herein by reference, and in particular see VH, VL, HCDR1-3, and LCDR1-3, scDB, scFV, and Fab sequences of SEQ ID NOs 1-12, 15-49, and 56-79, for CD16A and EGFR.
  • the poly specific binding molecule such as an antibody construct, of the present disclosure preferably comprises a binding domain, which specifically binds CD16A, comprising: a VL region comprising CDR-L1 as depicted in SEQ ID NO: 12, 21, 30, 38 or 44, a CDR-L2 as depicted in SEQ ID NO: 13, 22, 31, 39 or 45, and a CDR-L3 as depicted in SEQ ID NO: 14, 23, 32, 40 or 46, and a VH region comprising a CDR-H1 as depicted in SEQ ID NO: 9, 18, 27, 35 or XX41 a CDR-H2 as depicted in SEQ ID NO: 10, 19, 28, 36 or 42, and a CDR-H3 as depicted in SEQ ID NO: 11, 20, 29, 37 or 43.
  • a binding domain which specifically binds CD16A, comprising: a VL region comprising CDR-L1 as depicted in SEQ ID NO: 12, 21, 30, 38 or 44, a CDR-L2 as depict
  • the poly specific binding molecule such as an antibody construct, of the present disclosure preferably comprises a binding domain, which specifically binds CD16A, comprising: a VH region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that is identical to the sequence depicted in SEQ ID NO: 15, 24, 33, 47, 49, 51, 53, 55 or 57 and/or a VL region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that is identical to the sequence depicted in SEQ ID NO: 16, 25, 34, 48, 50, 52, 54, 56 or 58.
  • the poly specific binding molecule, such as an antibody construct, of the present disclosure may comprise a binding domain, which specifically binds EGFR, comprising: a VL region comprising CDR-L1 as depicted in SEQ ID NO: 62, a CDR-L2 as depicted in SEQ ID NO: 63, and a CDR-L3 as depicted in SEQ ID NO: 64, and a VH region comprising a CDR- H1 as depicted in SEQ ID NO: 59, a CDR-H2 as depicted in SEQ ID NO: 60, and a CDR-H3 as depicted in SEQ ID NO: 61.
  • the polyspecific binding molecule such as an antibody construct, of the present disclosure may comprise a binding domain, which specifically binds EGFR, comprising: a VH region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that is identical to the sequence depicted in SEQ ID NO: 65, and/ or a VL region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that is identical to the sequence depicted in SEQ ID NO: 66.
  • the poly specific binding molecule, such as an antibody construct, of the present disclosure may comprise a binding domain, which specifically binds FOLR1, comprising: a VL region comprising CDR-L1 as depicted in SEQ ID NO: 76 or 86, a CDR-L2 as depicted in SEQ ID NO: 77 or 87, and a CDR-L3 as depicted in SEQ ID NO: 78 or 88, and a VH region comprising a CDR-H1 as depicted in SEQ ID NO: 73 or 83, a CDR-H2 as depicted in SEQ ID NO: 74 or 84, and a CDR-H3 as depicted in SEQ ID NO: 75 or 85.
  • the polyspecific binding molecule such as an antibody construct, of the present disclosure may comprise a binding domain, which specifically binds FOLR1, comprising: a VH region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that is identical to the sequence depicted in SEQ ID NO: 79 or 89, and/ or a VL region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that is identical to the sequence depicted in SEQ ID NO: 80 or 90.
  • the polyspecific binding molecule, such as an antibody construct, of the present disclosure comprises amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or being identical to SEQ ID NOs: 67 and 68 or 69 and 70.
  • the polyspecific binding molecule, such as an antibody construct, of the present disclosure has two polypeptide chains, wherein the two polypeptide chains comprise or consist of the amino acid sequences set forth in SEQ ID NOs:67 and 68 or 69 and 70, preferably the amino acid sequences set forth in SEQ ID NOs: 67 and 68.
  • the polyspecific binding molecule such as an antibody construct of the present disclosure comprises amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or being identical to SEQ ID NOs: 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, or 103 and 104.
  • the polyspecific binding molecule such as an antibody construct of the present disclosure has two polypeptide chains, wherein the two polypeptide chains comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, or 103 and 104, preferably the amino acid sequences set forth in SEQ ID NOs: 101 and 102.
  • an immune checkpoint inhibitor is administered during the period of administration of the polyspecific binding molecule.
  • an immune checkpoint inhibitor is administered after the period of administration of the poly specific binding molecule, and after cells have been resensitized.
  • the ICI that is administered can be one that binds to CTLA-4, PD-1, or PD-L receptors on the surface of T-lymphocytes, and allowing activation of the T-lymphocytes towards the tumor.
  • the method of the disclosure uses an ICI that targets PD-1/PD-L1, which are immune-checkpoint molecules that inhibit the activation of antigen-presenting cells and T cells (priming phase) and the cytotoxic functions of T cells (effector phase).
  • Nivolumab (OPDIVOTM, Bristol Myers Squibb) is a human IgG4 monoclonal antibody that blocks PD-1. See W02006121168A1 (Ono Pharmaceutical Co. LTD.). Atezolizumab (TecentriqTM, Genetech) It is a humanized, monoclonal antibody of IgGl isotype against PD-L1 and is used for the treatment of various cancer types. See W02010077634A1 (Genetech). Pembrolizumab (KeytrudaTM, Merck) , is a humanized IgG4 isotype anti antibody against the PD-1 receptor. See WO2008156712A1 (N. V.
  • Cemiplimab (LibtayoTM, Regeneron Pharmaceuticals, Inc.) is a monoclonal antibody that binds to PD-1. See WO2015112800A1, H4H7798N (Regeneron Pharmaceuticals, Inc).
  • Dostarlimab (TSR-042, JEMPERLITM, Glaxo Smith Kline) is an humanized, monoclonal antibody of the IgG 4K isotype that binds to PD-1.
  • Durvalumab (ImfinziTM, Medimmune/AstraZeneca) is a human immunoglobulin G1 kappa (IgGlK) monoclonal antibody that blocks the interaction of PD-L1 with PD-1.
  • anti-PD-Ll antibodies include H2M8314N, avelumab, MDX-1105, LY3300054, FAZ053, STI-1014, CX-072, KN035, and CK-301.
  • immune effector cell may refer to any leukocyte or precursor involved e.g. in defending the body against cancer, diseases induced by infectious agents, foreign materials or autoimmune reactions.
  • the immune effector cells comprise B lymphocytes (B cells), T lymphocytes (T cells, including CD4+ and CD8+ T cells), NK cells, NKT cells, monocytes, macrophages, dendritic cells, mast cells, granulocytes such as neutrophils, basophils and eosinophils, innate lymphoid cells (ILCs, which comprise ILC-1, ILC-2 and ILC-3) or any combinations thereof.
  • the term immune effector cell refers to an NK cell, an ILC-1 cell, a NKT cell, a macrophage, a monocyte, and/or a T cell, such as a CD8+ T cell or a y6 T cell.
  • NK cells are CD56 + CD3 large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53 : 1666-1676). Unlike cytotoxic CD8+ T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization and can also eradicate MHC-I-negative cells (Nami- Mancinelli E, et al. Int Immunol 2011 23 : 427-431). NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms (Morgan R A, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter D L, et al. N Engl J Med 2011 365:725- 733), and on-target, off-tumor effects.
  • Macrophages are potent effectors of the innate immune system and are capable of at least three distinct anti-tumor functions: phagocytosis, cellular cytotoxicity, and antigen presentation to orchestrate an adaptive immune response. While T cells require antigendependent activation via the T cell receptor or the chimeric immunoreceptor, macrophages can be activated in a variety of ways. Direct macrophage activation is antigen-independent, relying on mechanisms such as pathogen associated molecular pattern recognition by Toll-like receptors (TLRs). Immune-complex mediated activation is antigen dependent but requires the presence of antigen- specific antibodies and absence of the inhibitory CD47-SIRPa interaction.
  • TLRs Toll-like receptors
  • T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate a different type of immune response.
  • APCs antigen-presenting cells
  • Cytotoxic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.
  • Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon reexposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.
  • Treg cells Regulatory T cells
  • suppressor T cells are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.
  • CD4+ Treg cells Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.
  • Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system.
  • NKT natural killer T
  • MHC major histocompatibility complex
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • Treatment includes the application or administration of the formulation to the body, an isolated tissue, or cell from a patient who has a disease/disorder, a symptom of a disease/disorder, or a predisposition toward a disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease.
  • Treatment can include administration of the poly specific binding molecule (PBM) by itself, such as in embodiments wherein the method re-sensitizes a subject to immune checkpoint inhibitor cancer treatment.
  • another cancer therapy drug can be given to the patient having the re-sensitized cells, such as and ICI.
  • Treatment can also include administering the PBM with another drug, particularly and ICI, during a treatment period.
  • the poly specific binding molecule is administered to the subject once every about 6 to about 8 days, or once every about 7 days. In some modes of treatment, wherein the polyspecific binding molecule is administered at a dose in the range of 7-8 mg/kg. In some modes of treatment, the PD-L1 inhibitor is administered to the subject once every about 13 to about 15 days, or once every about 14 days. In some modes of treatment, the PD-L1 inhibitor is administered at a dose in the range of 14-16 mg/kg.
  • the poly specific binding molecule and the PD-L1 inhibitor are administered to the subject at the same time, or about the same time.
  • the PD-L1 inhibitor is concurrently administered with the polyspecific binding molecule, and the PD-L1 inhibitor is present in an amount greater than the polyspecific binding molecule.
  • administering is carried out for a period of time in the range of two weeks to one year, or one month to ten months, or three months to nine months.
  • amelioration refers to any improvement of the disease state of a patient having a tumor or cancer or a metastatic cancer as defined elsewhere herein, by the administration of an antibody construct according to the invention to a subject in need thereof. Such an improvement may also be seen as a slowing or stopping of the progression of the tumor or cancer or metastatic cancer of the patient.
  • tumorous diseases or “tumor disease” refers to a disease characterized by the presence or development of a tumor.
  • a “tumor” is an abnormal growth of cells that serves no purpose. Tumors are divided into benign tumors, i.e. non-malignant tumors, and malignant tumors, i.e. cancerous tumors/cancer. While benign tumors grow slowly, have distinct borders and do not invade nearby tissue/do not spread to other parts of the body, malignant tumor can grow quickly, have irregular borders, often invade surrounding tissue and spread to other parts of the body called metastasis (Patel, JAMA Oncol, 2020, 6(9): 1488).
  • Tumors of the hematopoietic and lymphoid tissues are tumors that affect the blood, bone marrow, lymph, and lymphatic system (Vardiman et al.; Blood, 2009, 114(5): 937-51/ [0123] “ Solid tumors” refer to new growths of tissue, i.e. an abnormal mass of tissues, that usually does not contain cysts or liquid areas. These can occur anywhere in the body. Solid tumors may be benign (not cancer), or malignant (cancer). One speaks of benign tumors when tumors do not grow through (infiltrate) the surrounding tissue and do not form secondary tumors (metastases). Malignant solid tumors, on the other hand, destroy surrounding tissue and can spread to other parts of the body.
  • Malignant neoplasms are also known as cancer. It is particularly envisaged that “solid tumors” in the context of the present invention address malignant solid tumors, selected from the group consisting of brain, cancer, head and neck cancer, lung cancer, esophageal cancer, gastric cancer, hepatocellular c arcinoma, small intestine cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian c ancer, cervical cancer, endometrial cancer, prostate cancer, renal cancer, bladder cancer, thyro id cancer, skin cancer, melanoma, and sarcoma, preferably ovarian, breast, renal, lung, colorectal, and brain cancer.
  • malignant solid tumors selected from the group consisting of brain, cancer, head and neck cancer, lung cancer, esophageal cancer, gastric cancer, hepatocellular c arcinoma, small intestine cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian c ancer
  • Neoplasm is an abnormal growth of tissue, usually but not always forming a mass. When also forming a mass, it is commonly referred to as a “tumor”.
  • Neoplasms or tumors can be benign, potentially malignant (pre- cancerous), or malignant.
  • Malignant neoplasms are commonly called cancer. They usually invade and destroy the surrounding tissue and may form metastases, i.e., they spread to other parts, tissues or organs of the body.
  • metastases i.e., they spread to other parts, tissues or organs of the body.
  • the term “metastatic cancer” encompasses metastases to other tissues or organs than the one of the original tumor.
  • Lymphomas and leukemias are lymphoid neoplasms. For the purposes of the present invention, they are also encompassed by the terms “tumor” or “cancer”.
  • Proliferating diseases are characterized by an excessive proliferation of cells and turnover of cellular matrix as described e.g. in Sporn and Harris, The American Journal of Medicine, 1981, 70(6): 1231-1236.
  • a “hot” or “warm” tumor refers to a tumor having a T-cell-inflamed phenotype. Hot tumors often have many molecules on their surface that allow T cells to attack and kill the tumor cells. Such tumors show signs of inflammation, meaning the tumor has already been infiltrated by T cells to fight the cancerous cells. Hot tumors are likely to trigger a strong immune response and are likely to respond to immunotherapy.
  • a “cold tumor” refers to a tumor having a non-T-cell-inflamed phenotype, which has low T cell infiltration, or which have not been infiltrated with T cells.
  • Cold tumors tend to be surrounded by cells that are able to suppress the immune response and keep T cells from attacking the tumor cells and killing them. Due to the lack of T cells, it difficult to trigger an immune response with immunotherapy drugs in such tumors. Cold tumors usually display little response to immunotherapy. The difference between hot tumors and cold tumors are described in detail in Gajewski et al. (2017) Adv Exp Med Biol. 1036: 19- 31 and Maleki Vareki (2016) Journal for ImmunoTherapy of Cancer 6: 157.
  • subject in need or those “in need of treatment” includes those already with the disorder or disease, as well as those in which the disorder or disease is to be prevented.
  • subject in need or patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • composition relates to a composition which is suitable for administration to a patient, preferably a human patient.
  • the particularly preferred pharmaceutical composition of this invention comprises one or a plurality of the antibody con struct! s) of the invention, preferably in a therapeutically effective dose.
  • the pharmaceutical composition further comprises suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and/or adjuvants.
  • Acceptable constituents of the composition are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen, and lyophilized compositions.
  • “Pharmaceutically acceptable carrier” means any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers, e.g. phosphate buffered saline (PBS) solutions, water, suspensions, emulsions, such as oil/water emulsions, various types of wetting agents, liposomes, dispersion media and coatings, which are compatible with pharmaceutical administration, in particular with parenteral administration.
  • PBS phosphate buffered saline
  • emulsions such as oil/water emulsions
  • various types of wetting agents, liposomes, dispersion media and coatings which are compatible with pharmaceutical administration, in particular with parenteral administration.
  • the use of such media and agents in pharmaceutical compositions is well known in the art, and the compositions comprising such carriers can be formulated by well-known conventional methods.
  • the term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect.
  • therapeutically effective dose is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts or doses effective for this use will depend on the condition to be treated (the indication), the delivered antibody construct, the therapeutic context and objectives, the severity of the disease, prior therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient, and the general state of the patient's own immune system. The proper dose can be adjusted according to the judgment of the attending physician such that it can be administered to the patient once or over a series of administrations, and in order to obtain the optimal therapeutic effect.
  • kit means two or more components - one of which corresponding to the antibody construct, the pharmaceutical composition, the vector or the host cell of the invention - packaged together in a container, recipient or otherwise.
  • a kit can hence be described as a set of products and/or utensils that are sufficient to achieve a certain goal, which can be marketed as a single unit.
  • compositions comprising the antibody construct defined in the context of the invention and further one or more excipients such as those illustratively described in this section and elsewhere herein.
  • Excipients can be used in the invention in this regard for a wide variety of purposes, such as adjusting physical, chemical, or biological properties of formulations, such as adjustment of viscosity, and or processes of one aspect of the invention to improve effectiveness and or to stabilize such formulations and processes against degradation and spoilage due to, for instance, stresses that occur during manufacturing, shipping, storage, pre-use preparation, administration, and thereafter.
  • the pharmaceutical composition may contain formulation materials for the purpose of modifying, maintaining or preserving, e.g., the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition (see, REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company).
  • formulation materials for the purpose of modifying, maintaining or preserving, e.g., the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition (see, REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company).
  • suitable formulation materials may include, but are not limited to: amino acids such as glycine, alanine, glutamine, asparagine, threonine, proline, 2- phenylalanine, including charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and/or histidine; antimicrobials such as antibacterial and antifungal agents; antioxidants such as ascorbic acid, methionine, sodium sulfite or sodium hydrogen- sulfite; buffers, buffer systems and buffering agents which are used to maintain the composition at physiological pH or at a slightly lower pH; examples of buffers are borate, bicarbonate; Tris-HCI, citrates, phosphates or other organic acids, succinate, phosphate, and histidine; for example Tris buffer of about pH 7.0-8.5; non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as e
  • amino acids
  • amino acid can act as a buffer, a stabilizer and/or an antioxidant
  • mannitol can act as a bulking agent and/or a tonicity enhancing agent
  • sodium chloride can act as delivery vehicle and/or tonicity enhancing agent; etc.
  • composition of the invention might comprise, in addition to the polypeptide of the invention defined herein, further biologically active agents, depending on the intended use of the composition.
  • the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra.
  • a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving the antibody construct of the invention in sustained- or controlled-delivery / release formulations.
  • the antibody construct may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatine-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be accomplished by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution.
  • Compositions for parenteral administration can be stored in lyophilized form or in a solution. Parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • composition is administered to a patient intravenously.
  • said tumorous disease is a solid tumor.
  • Solid tumors or cancer comprise but are not limited to breast cancer (BC), Colorectal cancer (CRC), Non-small-cell lung carcinoma (NSCLC), Small-cell carcinoma (SCLC also known as "small-cell lung cancer", or “oat-cell carcinoma"), Prostate cancer (PC), Glioblastoma (also known as glioblastoma multiforme (GBM)).
  • said tumorous disease is a metastatic tumor
  • the present invention also provides a method for the treatment or amelioration of a disease, the method comprising the step of administering to a subject in need thereof an antibody construct according to the invention.
  • the antibody construct of the invention will generally be designed for specific routes and methods of administration, for specific dosages and frequencies of administration, for specific treatments of specific diseases, with ranges of bio-availability and persistence, among other things.
  • the materials of the composition are preferably formulated in concentrations that are acceptable for the site of administration.
  • the present invention provides for an uninterrupted administration of the suitable composition.
  • uninterrupted or substantially uninterrupted, i.e. continuous administration may be realized by a small pump system worn by the patient for metering the influx of therapeutic agent into the body of the patient.
  • the pharmaceutical composition comprising the antibody construct of the invention can be administered by using said pump systems.
  • Such pump systems are generally known in the art, and commonly rely on periodic exchange of cartridges containing the therapeutic agent to be infused.
  • a temporary interruption of the otherwise uninterrupted flow of therapeutic agent into the body of the patient may ensue.
  • the phase of administration prior to cartridge replacement and the phase of administration following cartridge replacement would still be considered within the meaning of the pharmaceutical means and methods of the invention together make up one “uninterrupted administration” of such therapeutic agent.
  • the lyophilized material is first reconstituted in an appropriate liquid prior to administration.
  • the lyophilized material may be reconstituted in, e.g., bacteriostatic water for injection (BWFI), physiological saline, phosphate buffered saline (PBS), or the same formulation the protein had been in prior to lyophilization.
  • BWFI bacteriostatic water for injection
  • PBS phosphate buffered saline
  • a therapeutic effective amount or dosage of an antibody construct of the invention preferably results in a decrease in severity of disease symptoms, an increase in frequency or duration of disease symptom-free periods or a prevention of impairment or disability due to the disease affliction.
  • a therapeutically effective amount of the antibody construct of the invention preferably inhibits cell growth or tumor growth by at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% relative to untreated patients.
  • the ability of a compound to inhibit tumor growth may be evaluated in an animal model predictive of efficacy in human tumors.
  • the present invention also relates to a kit comprising an antibody construct of the invention, a nucleic acid molecule of the invention, a vector of the invention or a host cell of the invention.
  • the kit may comprise one or more recipients (such as vials, ampoules, containers, syringes, bottles, bags) of any appropriate shape, size and material (preferably waterproof, e.g. plastic or glass) containing the antibody construct or the pharmaceutical composition of the present invention in an appropriate dosage for administration.
  • the kit may additionally contain instructions for use (e.g.
  • kits for a single-dose administration unit may also contain a first recipient comprising a dried / lyophilized antibody construct and a second recipient comprising an aqueous formulation.
  • kits containing single-chambered and multi-chambered pre-filled syringes are provided.
  • the kit of the invention may typically comprise a container comprising the antibody construct of the invention, the nucleic acid molecule of the invention, the vector of the invention, or the host cell of the invention, and optionally one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a reagent includes one or more of such different reagents
  • reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
  • the term “about” or “approximately” as used herein means within 10%, preferably within 5%, more preferably within 2%, even more preferably within 1% of a given value or range (plus (+) or minus (-)). It includes, however, also the concrete number, e.g., about 20 includes 20.
  • the term “less than” or “greater than” includes the concrete number. For example, less than 20 means less than or equal to. Similarly, more than or greater than means more than or equal to, or greater than or equal to, respectively.
  • Example 1 Effect of an CD16/EGFR specific Innate Cell Engager on tumor immunology
  • EGFR is frequently overexpressed on the cell surface in solid tumors, and is associated with poor prognosis. Moreover, it has been described that some patients do not respond to EGFR inhibitors, and in patients that do, acquired resistance invariably occurs; novel therapies acting independently of EGFR signaling are required. For the present study an CD16/EGFR bispecific ICE® was administered to patients with advanced/metastatic solid malignancies known to express EGFR (Study AFM24-101/NCT04259450).
  • Cytokines are soluble proteins which are released by immune cells and which exert effects on other cells.
  • the studied cytokines are either typically released by NK cells and other immune cells during inflammation, or they can have effects on the studied effector cells or both. Cytokines were quantified in plasma samples isolated from peripheral blood, thus, the majority of cytokine release results from effects on immune cells circulating within the bloodstream. However, cytokines released from tissues are also taken up by the bloodstream and can result in low but measurable increase in cytokine levels in plasma.
  • Pre-dose levels of IL2, IL6, and IL15 did not show an increase correlated to higher doses of AFM24, as they are not secreted by AFM24 target cells.
  • IL6 was analyzed as acute safety assessment, IL2 and IL 15 were analyzed as they have direct effects on AFM24 target cells.
  • IL2 concentrations were at the LLOQ for most of the patients across all time points measured.
  • Immunophenotyping allows the analysis of immune cells in peripheral blood. Relative amounts of immune cell subtypes and activation status of the cells are analyzed. Peripheral Blood Mononuclear Cells were collected before start of treatment and at several timepoints later on to track the changes in the activation status of the effector cells as well as for the absolute and relative baseline number of cells. [0174] AFM24 exerts its effect over linking and engaging NK cells or macrophages to target EGFR expressing cells. Thus, without these effector cells, the activity of AFM24 might be limited. Therefore, a certain number of NK cells is needed to enable tumor cell killing mediated by AFM24.
  • NK cells are mainly based in tissue
  • peripheral blood only absolute NK cell numbers were assessed.
  • patients showed mean baseline levels of 95-202 NK cells / pl of blood as calculated from the relative number of NK cells / lymphocytes vs lymphocyte levels from differential blood analysis.
  • CD16A is also known as FcyRIII and the receptor leading to ADCC when being engaged over target cell.
  • CD16A downregulation can be considered as a functional activation marker, since antibody binding to the target (tumor) cell and activating the effector cell (NK cell) through CD16A lead to ADCC and consequently can result in shedding of the CD16A receptor.
  • CD16Ahigh NK cells partially extravasate after activation and binding to AFM24, thus leading to a relative reduction of CD 16 A.
  • CD69 and Ki-67 are accepted activation markers on immune cells. Both markers are upregulated on NK cells after AFM24 administration. NK cells become activated by AFM24 already at lower doses and immediately after the first dosing of AFM24 ( Figure 2, see graph B for Ki-67, CD69 see Berrien-Elliott MM, et al. Cancer Discov 2020;10(12): 1854-71.). It was observed in the study that AFM24 treatment leads to an indirect activation of T cells. T cell activation is more pronounced at higher doses and occurs later during AFM24 treatment compared to NK cell activation ( Figure 2, see graphs D and E). This is in line with the physiological time the immune system needs to activate the adaptive immune system.
  • Biopsies were taken at screening and at day 24 of treatment, although for operational reasons, deviations of several days were accepted.
  • Immunohistochemistry allows the identification of antigens/cellular markers in certain areas of the biopsy. As the architecture of the biopsy section is preserved in histological analyses, cells can be attributed to be located in the tumor, even if the biopsy contains tumor and healthy tissue. Quantification of markers in IHC was done for the tumor area only.
  • Tumor content ranged from 0% (in two cases) to almost 75%. Biopsies containing no tumor tissue were excluded from further analyses.
  • Paired tumor biopsies could be obtained from 18 of the 35 treated patients. Of these 18 paired biopsies, only the biopsies were analyzed further which contained tumor tissue (16 pairs).
  • Tumor samples were analyzed for the expression of EGFR, CD3, CD8, CD45, CD56, CD68, and CD 163 by IHC.
  • CD3 positive (CD3+) cell density was determined by image analysis using the VisiopharmTM software. Except for the clinically validated EGFR assessment, the analyses were not validated and exploratory.
  • EGFR expression was quantified by a board-certified pathologist using the H-score obtained by the formula: 3* percentage of strongly staining nuclei + 2/ percentage of moderately staining nuclei + percentage of weakly staining nuclei, giving a range of 0 to 300.
  • H-Score which is calculated using low, medium and high intensity staining and the respective positive area of interest.
  • NK cells (as stained with anti-CD56) could not be reliably quantified due to background signals, thus, NK cell numbers are not reported here.
  • T cells (as stained with anti- CD3) increased in the biopsies of most patients, especially in doses of 160 mg and above (see Figure 5, parts A and B).
  • Biopsies from patients who received the higher doses of AFM24 had additional gene expression profiling (C1D24 versus screening).
  • the assay was not validated and exploratory. 250 ng of total RNA quantified using the NanoDropTM 2000 (Thermo Scientific), were directly hybridized (at 65°C for 18 hours) with the nCounter® PanCancer Immune Profiling Panel and the nCounter® Tumor Signaling 360TM panel following manufacturer’s instructions. After solution-phase hybridization between target RNA and reporter-capture probe pairs, excess probes were washed away using a two steps magnetic bead-based purification on the nCounter® Prep Station.
  • RNA / Probe complexes were aligned and immobilized in the cartridge for data collection.
  • the cartridge was then transferred to the nCounter® Digital Analyzer for image acquisition and counts collection. Quality control was done according to default settings. Background correction was conducted with background thresholding by calculating the mean of negative control expression plus double of the standard deviation.
  • the expression counts were normalized using the most stable housekeeping genes using the geNorm algorithm according to default settings.
  • Cell type scores were calculated as the geometric mean of log2 normalized expression levels of cell type-specific genes. Differentially expressed genes were identified using the optimal workflow of the nCounter® Advanced Analysis module with Bejamini -Hochberg p-value adjustment and patient ID as a confounder.
  • Pathway scores have been calculated by pathway level analysis of gene expression (PLAGE) as implemented in the nCounter® Advanced Analysis module. Visualization of results has been done using R v4.3.0.
  • Non-spatial expression profiling was performed to detect changes in cellular composition and signaling in tumor biopsies which can be linked to the treatment of AFM24 and which could not be stained by immunohistochemistry.
  • Example 2 Case studies of patients treated with AFM24-102
  • AFM24-102 (Study NCT05109442) is a Phase l/2a open-label, non-randomized, multicenter, dose escalation, and expansion study evaluating AFM24 in combination with atezolizumab in patients with selected EGRF-expressing advanced solid malignancies whose disease has progressed after treatment with previous anticancer therapies.
  • phase 1 There are 2 parts in this study: a dose escalation phase (phase 1) and an expansion phase (phase 2a). Patients are qualified to receive the investigational drugs (AFM24 + atezolizumab) in the dose escalation phase or the expansion phase only if they are deemed eligible following the safety lead-in phase. Seven days before the planned first combination treatment, patients receive a single dose of AFM24 and are observed for any adverse events for 1 week.
  • phase 1 a dose escalation phase
  • phase 2a an expansion phase
  • the dose escalation phase was followed by the expansion phase once the recommended phase 2 dose (RP2D) of AFM24 in combination with atezolizumab has been determined.
  • the administration schema is depicted in Figure 7, parts A and 7.
  • the expansion phase of the study is intended to collect preliminary evidence of efficacy and to further confirm the safety of AFM24 in combination with atezolizumab.
  • Atezolizumab monotherapy after progression on a CPI has very limited activity, with an ORR of 0-7% and a median PFS of 2.9 to 3.9 months (Fujita, 2019 Thorac Cancer. 2020 11(1): 15-18; Kitagawa, 2020 Thorac Cancer. 2020 11(7): 1927-1933); hence, it is unlikely that the results observed so far in NSCLC EGFRwt cohort are driven solely by atezolizumab, but that they are potentially the result of the combination of AFM24 and atezolizumab in this population.
  • AFM24 with atezolizumab is well tolerated, toxicities are manageable, with no new or unexpected toxicities observed compared to the safety profile of each single agent.
  • most of the patients enrolled in the study have been heavily pretreated including cytotoxic agents and consequently may have experienced considerable toxicities. Therefore, a tolerable treatment option is desirable in particular in this setting.
  • Atezolizumab receives weekly infusions of AFM24; the first administration of AFM24 is given as a single drug to assess its tolerability seven days prior to Cycle 1 Day 1 (i.e., at Day - 7).
  • the subsequent administration consists of the application of atezolizumab within 60 minutes, followed by the per-protocol mandated premedication (i.e., dexamethasone, Hl antagonist with or without H2 antagonist and oral acetaminophen) within 60 minutes.
  • AFM24 infusion starts.
  • a fixed dose of 480 mg of AFM24 is given weekly, and 840 mg of atezolizumab are given every two weeks (which is one of the approved recommended dose regimens for the treatment of NSCLC). If the infusions in Cycle 1 are well tolerated, the premedication regimen can be tapered/decreased.
  • Treatment is given in four-week cycles until disease progression, intolerable toxicity, investigator discretion, or patient withdrawal of consent. Tumor assessments are performed at screening, cycles 2, 4, 6, 8, 10, 12; and every three cycles thereafter.
  • CTR consolidative thoracic radiation therapy
  • stage IIIB 66-year-old male diagnosed in April 2022 as stage IIIB.
  • stage IV 55-year-old male, diagnosed in Mar 2022 as stage IV of Tubular Mucinous metastatic Gastric cancer, PD-L1 neg, TP53 mut, PIK3CA mut, APC mut and SMAD4 mut.
  • Antibody -induced release of inflammatory cytokines was assessed in cultures of human PBMC from healthy donors in the presence and absence of target antigen-expressing tumor cells. PBMCs of five individual donors were incubated with or without A-431 tumor target cells and increasing concentrations of AFM24, control or comparator antibodies. As a positive control, T cell activator beads (CD3/CD28 DynaBeads) stimulating a T cell response in vitro were included. The release of IL-6, TNF-a, and IFN-y into the cell culture supernatant was quantified after 4h (see Figure 16)
  • CD3/CD28 DynaBeads stimulated the release of all tested cytokines after 4h verifying the experimental setup. No or only marginal release of all analyzed cytokines was detected upon stimulation with AFM24 for 4h in the absence of A-431 target cells. In contrast, in the presence of A-431 target cells at an target ratio of 50: 1, AFM24 stimulated the release of IL- 6, TNF-a and IFN-y in a concentration-dependent manner. After 4h incubation with AFM24, a modest release of IL-6 was measured.
  • cytokines IL-6, TNF-a and IFN- y were strictly dependent on the presence of both targeting specificities incorporated in AFM24, the anti-CD16A and the anti -EGFR domains, as demonstrated by absent cytokine release upon incubation with the control antibodies scFv-IgAb_44 (anti-RSV/CD16A) and scFv-IgAb_45 (anti-EGFR/RSV).
  • cytokine concentrations observed with T cell activator beads were substantially higher than in presence of AFM24, suggesting that engagement of NK cells for antibody-mediated target cell lysis is associated with low level cytokine release when compared with T cell activation in cultures of human PBMC.
  • T cell activation but not stimulation with AFM24 induced release of IL-2, IL-4, and IL- 10, suggesting that AFM24- induced NK cell activation neither directly nor indirectly stimulated release of these cytokines in human PBMC under the experimental setting used.
  • Antibody -induced release of inflammatory cytokines was assessed in cultures of human PBMC from healthy donors in the presence and absence of target antigen-expressing tumor cells. PBMCs of five individual donors were incubated with or without OVCAR-3 tumor target cells and AFM32, control or comparator antibodies. The release of IL-6, TNF-a, and IFN-y into the cell culture supernatant was quantified after 4h (see Figure 17) [0277] AFM32 enhanced the release of IL-6 in the absence of target cells during 4 h incubation in 2 out of 3 experiments (212 pg/mL & 174 pg/mL) as compared to without antibody (mean level: 26 pg/mL).
  • FRa-positive OVCAR-3 cells slightly stimulated antibody-independent release after 4 h incubation.
  • IL-6 release was induced by AFM32, by human IgGl anti-FRa IgAb_335, and by scFv-IgAb_162 (FRa/RSV). This effect was more pronounced after 24 h with IL-6 levels exceeding 1000 pg/mL for all three antibodies.
  • AFM32 In the absence of target cells, AFM32 induced the release of low levels of TNF-a after 4 h (mean: 63 pg/mL TNF- a at 100 pg/mL AFM32) relative to without antibody (mean: 15 pg/mL TNF- a.
  • TNF- a release was induced by AFM32 (mean: 90 pg/mL) and to a lower extent also by human IgGl anti-FR a IgAb_335 (mean: 26 pg/mL) and scFv-IgAb_162 FOLR1/RSV scFv-IgAb (mean: 16 pg/mL) after 24 h. A similar trend was observed after 4 h incubation.
  • AFM32 In the presence of FRa-positive target cells AFM32 induced IL-6, IL-10, TNF-a, and in a concentration-dependent manner IFN-y, which is associated with AFM32’s mechanism of action that involves crosslinking of CD16A-positive immune cells with FRa-positive target cells and subsequent activation of these immune cells.
  • the control item scFv-IgAb_444 did not induce substantial cytokine release in the absence or presence of target cells, suggesting that bivalent binding to CD16A+ cells is not sufficient to trigger cytokine release.
  • FRa/RSV scFv-IgAb_162 that is not known to bind to any immune cells in PBMC induced the release of IL-6 and IFN-y in presence of FRa-positive target cells, for so far unknown reasons.
  • the human anti-FRa IgGl (IgAb_335) induced in general the same cytokines as AFM32 but for most cytokines at lower levels which is most likely due to its lower apparent avidity for CD16A-positive cells. Incubation of PBMC with FRa-negative Raji cells led to antibody-independent cytokine release, most likely due to high sensitivity of target cells to natural cytotoxicity.
  • the positive control (CD3/CD28 activator beads) induced strong cytokine release of IL-2, IL-4, IL-6, IL-10, TNF-a, and IFN-y in all experiments.
  • ADCP antibody-dependent cellular phagocytosis
  • AFM24 and AFM32 were designed to interact with CD16A-expressing innate immune cells such as NK cells and macrophages and cancer cells in the tumor microenvironment with the intent of inducing an efficient anti-tumoral response.
  • CD16A-positive macrophages may serve as abundant effector cells.
  • the current investigations aimed to assess the ability of AFM24 and AFM32 in mediating an anti-tumoral response by engaging human macrophages.
  • ADCP Antibody-dependent cellular phagocytosis
  • AFM24 only induced phagocytosis of the EGFR-positive cell lines DK-MG and HCT- 116 (RAS mutation G13D), while EGFR-negative KARPAS-299 cells were unaffected, demonstrating strict antigen-specificity.
  • ADCP-induction towards DK-MG cells by AFM24 was comparable to ADCP induced by cetuximab (anti-EGFR IgGl) or other anti-EGFR IgGl antibodies harboring functional Fc-parts.
  • ADCP towards HCT-116 cells was exclusively detected in the presence of AFM24, while cetuximab and two other anti-EGFR IgGl antibodies did not induce target cell phagocytosis.
  • cetuximab and two other anti-EGFR IgGl antibodies did not induce target cell phagocytosis.
  • ADCP antibody-dependent cellular phagocytosis
  • AFM24 and AFM32 demonstrate similar in vivo anti-tumor control against target positive tumor cells
  • mice received either vehicle alone or NK cells (2.7 x 10 6 cells/mouse) in combination with vehicle, negative control (RSV/CD16A) or titrated AFM24 (co-administration), or NK cells pre-loaded with AFM24 for 1 hour followed by removal of excess AFM24. Thereafter, AFM24, RSV/CD16A or vehicle had been readministered once a week.
  • the administration schema is depicted in Figure 20 part A.
  • mice had received human IL-2 three times per week.
  • BLI bioluminescence imaging
  • hIL-15 human interleukin- 15
  • IL-2 interleukin-2
  • i.p. intraperitoneally
  • TNBC triple negative breast cancer
  • AFM24 in combination with adoptive NK cells, leads to dose-dependent tumor regression in a mouse xenograft model, see Figure 20, part B.
  • AFM32 treatment also resulted in significant reduction in terminal tumor weights.
  • Tumor weight inhibition (TWI) 41.11%, 47.31%, 46.52% and 46.55% compared with the vehicle control (mean ⁇ SEM, 0.459 ⁇ 0.024 g) were observed in groups treated with 3 mg/kg (0.271 ⁇ 0.011 g), 6 mg/kg (0.242 ⁇ 0.006 g), 30 mg/kg (0.246 ⁇ 0.019 g) and 60 mg/kg (0.246 ⁇ 0.015 g) of AFM32, respectively.
  • the difference between the vehicle control was significant with the AFM32 treatment at 6 mg/kg or higher (P ⁇ 0.0010, Kruskal -Wallis test with Dunn’s multiple comparisons test). BW changes were not different between the groups throughout the study, indicating all the treatments were well tolerated without unexpected clinical observations or deaths.

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Abstract

L'invention concerne des méthodes pour le traitement du cancer chez un sujet, le sujet étant, ou étant prédit comme étant, résistant au traitement du cancer par inhibiteur de point de contrôle immunitaire (ICI). Une molécule de liaison polyspécifique qui se lie à CD16A sur des cellules immunitaires innées et un antigène tumoral, tel que EGFR ou FOLR1, sur des cellules tumorales, est administrée au sujet résistant. La molécule de liaison polyspécifique peut sensibiliser la tumeur vis-à-vis d'un traitement avec un ICI, tel qu'un inhibiteur de PD-L1 ou de PD-1.
PCT/IB2024/055675 2024-06-10 2024-06-10 Liant polyspécifique d'antigène de cd16a/tumeur destiné à être utilisé dans le traitement de la résistance à un inhibiteur de point de contrôle immunitaire Pending WO2025257588A1 (fr)

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