CD16a / tumor antigen polyspecific binder for use in the treatment of immune checkpoint inhibitor resistance

Abstract

Methods are disclosed for the treatment of cancer in a subject, wherein the subject is, or is predicted to be, resistant to immune checkpoint inhibitor (ICI) cancer treatment. A polyspecific binding molecule that binds to CD16A on innate immune cells and a tumor antigen, such as EGFR or FOLR1, on tumor cells, is administered to the resistant subject. The polyspecific binding molecule can sensitize the tumor towards treatment with an ICI, such as a PD-L1 or PD-1 inhibitor.

Description

CD16A / TUM0R ANTIGEN POLYSPECIFIC BINDER FOR USE IN THE TREATMENT OF IMMUNE CHECKPOINT INHIBITOR RESISTANCEField of the Invention

[0001] 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.Background

[0002] Immune checkpoint inhibitors (ICIs) 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.

[0003] Some 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.

[0004] While many patients can obtain clinical benefits from ICI treatment, a large number of patients are primarily resistant to such treatment or acquire resistance after an initial response. There are various resistance mechanisms, such as insufficient antigen recognition by T cells,impaired T-cell migration and / or infiltration, and reduced T-cell cytotoxicity, most of which are related to the T-cell activation process.Summary

[0005] 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. 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. In this regard, 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. After the administration of a polyspecific biding molecule, an increase in NK cell, T cell and cytotoxicity associated functional genes, such as Granzyme, was observed, suggesting an increase in these cell functions upon treatment. In patients receiving this therapy, partial responses (PRs) and complete response (CRs) were seen, revealing that the disease had stabilized. It is believed that 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).

[0006] As such, use of the polyspecific binding molecule (e.g., anti-CD16A / anti-EGFR) binding molecule can in essence “transform” immune checkpoint inhibitor-resistant patients into immune checkpoint inhibitor-sensitive patients. Likewise, since factors that are predictive to resistance to immune checkpoint inhibitor cancer treatment are known, 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. In methods of the invention, 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.

[0007] Accordingly, in one aspect 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 checkpointinhibitor 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

[0008] In some case, the solid tumor is characterized by epidermal growth factor receptor (EGFR) overexpression on solid tumor cells, and the polyspecific binding molecule binds to EGFR. 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).

[0009] In some cases, 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).

[0010] 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. For example, 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. In some cases, the subject has previously undergone treatment with a non-biologic, such as a platinum-based therapeutic treatment, and a immune checkpoint inhibitor. In some cases, the subject has an actionable driver mutation other than an EGFR mutation, and has undergone prior treatment for the mutation. For example, 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.

[0011] In another aspect, 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.

[0012] In another aspect, 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.

[0013] During or after the method of treatment according to the invention, 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.Brief Description of the Drawings

[0014] 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%.

[0015] 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.

[0016] Figure 3: EGFR staining of paired biopsies.A) Line plot of individual biopsies H-Score measurements for EGFR expression. Different dose cohorts are separated by color.B) Representative image of EGFR staining in a NSCLC sample with 160 mg of AFM24 at screening.

[0017] 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.

[0018] 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.A: Box plot of all macrophages as stained with CD68 B: Box plot of M2 macrophages as stained with CD 163.

[0019] Figure 5: Gene expression profiling and IHC of biopsies indicate an increase in cytotoxic cells within the tumorA): Quantification of T cells in paired biopsies from screening and at C1D24. Cohorts were grouped by color. The red cross indicates the mean measurement.B): Immunohistochemistry was performed to analyze tumor content of immune cells.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.

[0020] Figure 6: Expression profiling cell type scoresTumor 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.A): Box plot showing log2 cell type score for CD56dim NK cells, n=10, log2 cell type score for CD8 T cells, n=10, and log2 cell type score for cytotoxic cells (as defined by the test kit), n=10.B): Genes associated using nCounter® Advanced Analysis module. Box plots showing the pathway activity score for NK cell functions, T cell functions, and cytotoxicity.

[0021] Figure 7: Treatment schedule in AFM24 clinical studies A: Study AFM24-101 (NCT04259450)B: Study AFM24-102 (NCT05109442) RP2D for AFM24 is weekly 480mg andAtezolizumab dose is every other week 840 mg

[0022] Figure 8: Waterfall Plot for Best %-Change from baseline in the Sum of Longest Diameters (Investigator’s Assessment). Evaluation per RECIST vl. l criteria.

[0023] 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.

[0024] 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.

[0025] Figure 11: A Patient exhibiting a Complete Response (CR) with AFM24 and Atezolizumab. Tumor Response by Investigator Assessment per RECIST vl.l. All non-target lesions are reported as absent. A shows TL1 (left para-aortic lymph node) and NTL2(retroperitoneal lymph node). B shows NTL1 (subcarinal lymph node). C shows NTL3 (right lower lobe septal thickening)

[0026] Figure 12: A Patient exhibiting a PR experienced a 50,5% overall shrinkage of their target lesions with AFM24 and Atezolizumab. Tumor Response by Investigator Assessment per RECIST vl .1. Initial response to treatment: Stable Disease. After 4 cycles of treatment: Unconfirmed Partial Response, confirmed after 6 cycles.

[0027] Figure 13: Response of a CPI-Refractory patient demonstrates the ability to rechallenge and synergize with CPI due to alleged reactivation of immune function by AFM24 administration. Gastric cancer patient showing Partial Response and ~ minus 70% tumor shrinkage in target lesions. Gastric cancer patient had previously progressed on 4 lines of therapy, including anti-PD-1 / chemotherapy combo. The patient’s skin metastases did not respond to any prior treatment but responded to AFM24 / atezolizumab combination therapy according to the invention.

[0028] 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.

[0029] Figure 15: A Patient exhibiting a PR experienced a 32% overall shrinkage of their target lesions with AFM24 and Atezolizumab. Patient was on SD until TA3 (-26.3% compared to baseline). Change in SOD compared to baseline: - 32%. Non-Target lesions were evaluated as present (without changes).

[0030] 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. Following 4h incubation, the levels of IL-6 (A), TNF-a (B), and IFN-y (C) were quantified in the cell culture supernatants.

[0031] Figure 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. Following co-incubation of PBMC, target cells and antibodies for 4 h, the levels of IL-6 , TNF-a , and IFN-y were quantified in the cell culture supernatants and plotted (samples with antibodies: mean values and standard deviations of 3independent experiments. Samples without antibodies: mean values and standard deviations of 3 independent experiments in technical duplicates).

[0032] Figure 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).

[0033] Figure 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.

[0034] Figure 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). As read out for the in vivo mouse model the median wholebody fluorescence was measured (B).

[0035] Figure 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 xlO6cells per mouse) were subcutaneously injected on Day 0. Animals were treated with either vehicle or AFM32 (3, 10, 30, or 60 mg / kg; IV, BIW) from Day 18 until the last observation day (Day 46). * 0.01<P<0.05 ** 0.001<P< 0.01 *** 0.0001<P<0.001 **** P< 0.0001. (C) Tumor volumes on Day 46 post-inoculation (Day 28 post-treatment initiation). The dotted line depicts the baseline TV. The data are presented as the mean ± SEM and each dot represents a TV of an individual animal on Day 46. SKOV3 Red FLuc cells (5 xlO6cells per mouse) were subcutaneously injected on Day 0. Animals were treated with either vehicle or AFM32 (3, 10, 30, or 60 mg / kg; IV, BIW) from Day 18 until the last observation day (Day 46).Detailed Description

[0036] This invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0037] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0038] 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. In the methods of the disclosure, _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.

[0039] 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. For example, 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.

[0040] In particular, 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. For example, after an initial does of the poly specific binding molecule, 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 subsequentdose(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. In other words, the response curve of cytokine production can flatten after the initial dose. Also observed was 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 Also, NK cells expressed the proliferation marker Ki-67, which is upregulated at later time point when cells enter the cell cycle. See, for example, Krzywinska, E. et al. (2015) EBioMedicine 2: 1364-1376. These increases in NK gene expression indicate NK cell engagement.

[0041] Also, the polyspecific binding molecule promotes re-activation of previously inactive T cells or engagement of further tumor-specific T cells. According to the disclosure, 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.

[0042] Preferably, subject that is treated has tumor that is characterized by epidermal growth factor receptor (EGFR) overexpression on the surface of the solid tumor cells. In turn the polyspecific binding molecule binds to EGFR on those tumor cells and also to CD16A on Natural Killer (NK) cells.

[0043] 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. For example, 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.

[0044] After the previous treatment, 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.

[0045] The previous ICI treatment can use an ICI that binds to immune inhibitory receptors, such as CTLA-4, PD-1, and PD-L receptors. For example, 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. See, for example, Lee, J.B., et al. (2022) Immune Checkpoint Inhibitors in 10 Years: Contribution of Basic Research and Clinical Application in Cancer Immunotherapy. Immune Netw. 22(l):e2.

[0046] The subject receiving previous treatment may have also been treated with one or more traditional chemotherapeutic drugs. For example, 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.

[0047] 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. 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. Also, taxanes are administered by lenghty IV infusions, which are difficult for many patients.

[0048] 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.

[0049] 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.

[0050] In other cases, 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).

[0051] 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. For example, the presence of PD-lhighCXCR5-TCFl- terminally differentiated exhausted T cells in the tumor microenvironment (TME), which are considered to be dysfunctional and not able to be reactivated using an ICI, could be determined. Also, 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.

[0052] . In some cases, the subject has an actionable driver mutation other than an EGFR mutation, and has undergone prior treatment for the mutation. For example, 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 (EGFR, ERBB2 (HER2), ALK, ROS1, RET, NTRK, MET, BRAF, and KRAS) have been associated with non-small cell lung cancer (NSCLC). 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.

[0053] 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. The term “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 targetsite 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.

[0054] The term “specifically binding”, as used herein means that the 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. In preferred embodiments, “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.

[0055] The term "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. An antibody construct is hence capable of binding to its specific target or antigen. Furthermore, 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. CDR1, CDR2 and CDR3 of the VL region) and the presence of a VH region comprising the three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). For the second binding domain to a tumor antigen, 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 constructsdefined in the context of the invention are based include for example monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies.

[0056] 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. Preferably, 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.

[0057] 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). 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.

[0058] 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.

[0059] As used herein, the terms "single-chain Fv," "single-chain antibodies" or "scFv" refer to single polypeptide chain antibody fragments that comprise the variable regions from both the heavy and light chains, but lack the constant regions. Generally, a single-chain antibody further comprises a polypeptide linker between the VH and VL domains which enables it toform the desired structure which would allow for antigen binding. Exemplary 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). Single chain antibodies are discussed in detail by Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods of generating single chain antibodies are known, including those described in U.S. Pat. Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242: 1038- 1041. In specific embodiments, 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. Generally, the arrangement of the VH and VL domains on the polypeptide chain within each of the scFv may be in any order. This means that 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. The term “double Fab” as used herein refers to two Fab fragments that are fused together, which are preferably staggered. Here, 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. As an illustrative example, the light chain-derived chain of the first Fab can be fused to the light chain derived-chain of the second Fab. As another illustrative example, the heavy chain- derived chain of the first Fab can be fused to the heavy chain derived-chain of the second Fab. As a further illustrative example, the heavy chain-derived chain of the first Fab can be fused to the light chain derived-chain of the second Fab. In some double Fabs, both chains of the two Fabs are fused together. For example, the light chain-derived chain of the first Fab can befused 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. Alternatively, 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.

[0060] 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.

[0061] 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.

[0062] As used herein, 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. Typically, 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. Preferred 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. Another preferred linker is (GGS)s. Accordingly, the VH and VL domains of one fragment are forced to pairwith the complementary VH and VL domains of another fragment, thereby forming two antigen-binding sites. A diabody can be formed by two separate polypeptide chains, each comprising a VH and a VL. Alternatively, all four variable domains can be comprised in one single polypeptide chain comprising two VH and two VL domains. In such a case, the diabody can also be termed “single chain diabody” or “scDb”. Typically, 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)?. On the polypeptide chain, 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. Similarly, 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. The term diabody does not exclude the fusion of further binding domains to the diabody.

[0063] In the context of the present invention, the definition of the term "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. For examples, 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. Moreover, the definition of the term "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). Examples for the above identified antibodies and variants or derivatives thereof are described inter alia in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Dubel, Antibody Engineering, Springer, 2nd ed. 2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0064] The term "bispecific" as used herein refers to an antibody construct which is "essentially bispecific", i.e., comprise specificities for two different antigens or targets, but nofurther specificity against a third or further antigen or target. Specifically, 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.

[0065] “ CD16A” or “CD 16a” 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. For example, 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.

[0066] “ 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. The amino acid sequence of human CD16B is given in UniProt entry 075015 (version 212 of 12 August 2020).

[0067] 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.

[0068] The term “CD16A shedding” or “shedding of CD16A” 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. “CD16A shedding” is known as activation-induced down-regulation as described e.g. in Romee at al., Blood, 2013, 121 (18):3599-3608), Peruzi et al., J. Immunol., 2013, 191 :955-957, Goodier et al., Front. Immunol., 2016, 7:384, andSrpan et al., J. Cell. Biol., 2018, 217(9):3267-3283, and the capacity of immune effector cells after CD16A shedding may then be impaired for several days.

[0069] The term "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.

[0070] Polyspecific binding molecules, and more particularly, bispecific binding molecules, that are used in methods of the disclosure, do not occur naturally and are markedly different from naturally occurring products. 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)

[0071] 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. This term thus preferably excludes sequences generated solely by genomic rearrangement in an immune cell in an animal. A "recombinant antibody" is an antibody made through the use of recombinant DNA technology or genetic engineering.

[0072] The term "monoclonal antibody" (mAb) or monoclonal antibody construct as used herein 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). In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridomaculture, 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.

[0073] 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 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 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.

[0074] 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). 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. Human germline sequences are disclosed e.g. in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, G.P. et al. (1995) Immunol.Today Vol. 16 (5): 237-242; and Tomlinson et al. (1995) EMBO J. 14: 14:4628- 4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequence, e.g., for framework regions and CDRs. Consensus human framework regions can also be used, for example as described in US Patent No. 6,300,064.

[0075] "Humanized" antibodies, antibody constructs, variants or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) are antibodies or immunoglobulins of mostly human sequences, which contain (a) minimal sequence(s) derived from non-human immunoglobulin. For the most part, 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. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, "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);Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593- 596 (1992).

[0076] 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.

[0077] 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.

[0078] 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).

[0079] The term "human antibody", "human antibody construct" and "human binding domain" 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. The definition of human antibodies, antibody constructs and binding domains as used herein, however, also contemplates "fully human antibodies", which include only non-artificially and / or genetically altered human sequences of antibodies as those can be derived by using technologies or systems such as the Xenomouse. Preferably, a "fully human antibody" does not include amino acid residues not encoded by human germline immunoglobulin sequences.

[0080] In some embodiments, 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.Preferably, 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. It is understood that 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. In preferred embodiments, 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. Ordinarily, however, an isolated antibody construct will be prepared by at least one purification step.

[0081] 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. Non-limiting embodiments of such 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. Strepll-tag) and His-tag. 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. Most preferably, a hexa-histidine tag is linked via peptide bond to the C-terminus of the antibody construct according to the invention. Additionally, a conjugate system of PLGA-PEG-PLGA may be combined with a polyhistidine tag for sustained release application and improved pharmacokinetic profile.

[0082] Amino acid sequence modifications of the antibody constructs which can be used in methods of the disclosure are also contemplated, as long as the minimal structural limitations of the first binding domain of the antibody construct of the present invention are maintained. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody construct. Amino acid sequence variants of the antibody constructs are prepared by introducing appropriate nucleotide changes into the antibody constructs nucleic acid, or by peptide synthesis. All of the below described amino acid sequence modifications should result in an antibody construct which still retains the desired biological activity (i.e. binding to CD16A, and / or the tumor cell surface antigen) of the unmodified parental molecule.

[0083] For amino acid sequences, 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. Preferably, 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.

[0084] An example of a useful algorithm is PILEUP. 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.

[0085] 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 etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, word threshold (T)=ll. 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.

[0086] An additional useful algorithm is gapped BLAST as reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. 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.

[0087] Generally, the 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%. In a similar manner, "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.

[0088] Generally, the 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%. Thus, 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.

[0089] The term “EGFR” refers to the epidermal growth factor receptor (EGFR; ErbB-1;HER1 in humans, including all isoforms or variants described with activation, mutations and implicated in pathophysiological processes. 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, TGFa, HB-EGF, neuregulins, betacellulin, amphiregulin) activates the intrinsic receptor tyrosine kinase in the intracellular domain through tyrosine phosphorylation and promotes receptor homo- or heterodimerization with HER family members. These intracellular phospho-tyrosines serve as docking sites for various adaptor proteins or enzymes including SHC, GRB2, PLCg and PI(3)K / Akt, which simultaneously initiate many signaling cascades that influence cell proliferation, angiogenesis, apoptosis resistance, invasion and metastasis. 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.

[0090] As used herein, the term “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. For example, 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). Due to its high expression in some tumors, FOLR1 is an attractive therapeutic target for the development of anti-cancer agents in order to limit toxic side-effectson off-target tissues. As used herein, “F0LR1” includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full length wild-type F0LR1.

[0091] As used herein, the term “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).

[0092] 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.

[0093] 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. Specific examples for 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)).

[0094] In some embodiments, 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.

[0095] In some embodiments, 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.

[0096] In some embodiments, 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.

[0097] In some embodiments, 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, HER4, FOLR1, and GD2) , as taught in W02023 / 007023. 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.

[0098] 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. 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.

[0099] 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.

[0100] 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.

[0101] In some embodiments, 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 beingidentical to SEQ ID NOs: 67 and 68 or 69 and 70. In some embodiments, 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.

[0102] In some embodiments, 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. In some embodiments, 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.

[0103] In embodiments of the disclosure, an immune checkpoint inhibitor (ICIs) is administered during the period of administration of the polyspecific binding molecule. In other embodiments of the disclosure, an immune checkpoint inhibitor (ICIs) is administered after the period of administration of the poly specific binding molecule, and after cells have been resensitized.

[0104] 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. In some embodiments, 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).

[0105] Nivolumab (OPDIVO™, Bristol Myers Squibb) is a human IgG4 monoclonal antibody that blocks PD-1. See W02006121168A1 (Ono Pharmaceutical Co. LTD.). Atezolizumab (Tecentriq™, 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 (Keytruda™, Merck) , is a humanized IgG4 isotype anti antibody against the PD-1 receptor. See WO2008156712A1 (N. V.Organon). Cemiplimab (Libtayo™, Regeneron Pharmaceuticals, Inc.) is a monoclonal antibody that binds to PD-1. See WO2015112800A1, H4H7798N (Regeneron Pharmaceuticals, Inc). Dostarlimab (TSR-042, JEMPERLI™, Glaxo Smith Kline) is anhumanized, monoclonal antibody of the IgG 4K isotype that binds to PD-1. Durvalumab (Imfinzi™, Medimmune / AstraZeneca) is a human immunoglobulin G1 kappa (IgGlK) monoclonal antibody that blocks the interaction of PD-L1 with PD-1.

[0106] Other anti-PD-1 antibodies include pidilizumab, MED10608, BI 754091, PF-0680159, spartalizumab, camrelizumab, JNJ-63723283, and MCLA-134.

[0107] Other anti-PD-Ll antibodies include H2M8314N, avelumab, MDX-1105, LY3300054, FAZ053, STI-1014, CX-072, KN035, and CK-301.

[0108] The term “immune effector cell” as used herein 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. For example, 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. Preferably, 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.

[0109] Natural killer (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.

[0110] Monocytes are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. They constitute between three to eight percent of the leukocytes in the blood. In the tissue monocytes mature into different types of macrophages at different anatomical locations. Monocytes have two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (approx.. 8-12 hours) to sites of infection in the tissues and divide / differentiate into macrophages anddendritic cells to elicit an immune response. Monocytes are usually identified in stained smears by their large bilobate nucleus.

[0111] 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.

[0112] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0113] T helper cells (TH 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.

[0114] Cytotoxic T cells (TC cells, or CTLs) 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.

[0115] 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” againstpast infections. Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0116] Regulatory T cells (Treg cells), formerly known as 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. Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.

[0117] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD Id.

[0118] The term "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. At a time after the PBM 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.

[0119] In some modes of treatment, 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.In some modes of treatment, the poly specific binding molecule and the PD-L1 inhibitor are administered to the subject at the same time, or about the same time. In some modes of treatment, 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. In some modes of treatment, administering is carried out for a period oftime in the range of two weeks to one year, or one month to ten months, or three months to nine months.

[0120] The term “amelioration” as used herein 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.

[0121] The term “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).

[0122] “ 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. This also includes those types of tumors described specifically in the context of EGFR and FOLR1 herein above. A “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 otherparts, tissues or organs of the body. Hence, 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”.

[0124] “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.

[0125] As used herein a “hot” or “warm” tumor, which is used interchangeably, 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.

[0126] As used herein, 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 (2018) Journal for ImmunoTherapy of Cancer 6: 157.

[0127] The terms “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. The subject in need or "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0128] The term “pharmaceutical 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. Preferably, 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. Pharmaceuticalcompositions of the invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0129] “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. 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.

[0130] The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “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.

[0131] The term “kit” as used herein 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.

[0132] Certain embodiments provide pharmaceutical 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 thatoccur during manufacturing, shipping, storage, pre-use preparation, administration, and thereafter.

[0133] In certain embodiments, 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). In such embodiments, 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 ethyl oleate; aqueous carriers including water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media; biodegradable polymers such as polyesters; bulking agents such as mannitol or glycine; chelating agents such as ethylenediamine tetraacetic acid (EDTA); isotonic and absorption delaying agents; complexing agents such as caffeine, polyvinylpyrrolidone, betacyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); carbohydrates may be nonreducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol or xylitol; (low molecular weight) proteins, polypeptides or proteinaceous carriers such as human or bovine serum albumin, gelatin or immunoglobulins, preferably of human origin; coloring and flavouring agents; sulfur containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thio sulfate; diluting agents; emulsifying agents; hydrophilic polymers such as polyvinylpyrrolidone); salt-forming counter-ions such as sodium; preservatives such as antimicrobials, anti-oxidants, chelating agents, inert gases and the like; examples are: benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); metal complexes such as Zn-protein complexes; solvents and cosolvents (such as glycerin, propylene glycol or polyethylene glycol); sugars and sugaralcohols, such as trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol stachyose, mannose, sorbose, xylose, ribose, myoinisitose, galactose, lactitol, ribitol, myoinisitol, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; and polyhydric sugar alcohols; suspending agents; surfactants or wetting agents such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal; surfactants may be detergents, preferably with a molecular weight of >1.2 KD and / or a polyether, preferably with a molecular weight of >3 KD; non-limiting examples for preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85; non-limiting examples for preferred polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG 5000; stability enhancing agents such as sucrose or sorbitol; tonicity enhancing agents such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol; parenteral delivery vehicles including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils; intravenous delivery vehicles including fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).

[0134] It is evident to those skilled in the art that the different constituents of the pharmaceutical composition (e.g., those listed above) can have different effects, for example, and 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.

[0135] It is envisaged that the 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.

[0136] In certain embodiments, 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. For example, 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.

[0137] 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. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticlesor porous beads and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. Sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 and European Patent Application Publication No. EP 058481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly (2-hydroxyethyl- methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D(-)-3- hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949.

[0138] 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. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0139] Pharmaceutical 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.

[0140] In one embodiment of the pharmaceutical composition according to one aspect of the invention the composition is administered to a patient intravenously.

[0141] Methods and protocols for the intravenous (iv) administration of pharmaceutical compositions described herein are well known in the art.

[0142] In one embodiment 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)).

[0143] In preferred embodiments said tumorous disease is a metastatic tumor

[0144] 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.

[0145] 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.

[0146] Formulations and compositions thus may be designed in accordance with the invention for delivery by any suitable route of administration. In the context of the present invention, the routes of administration include, but are not limited to topical routes (such as epicutaneous, inhalational, nasal, opthalmic, auricular / aural, vaginal, mucosal); enteral routes (such as oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and parenteral routes (such as intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extra-amniotic, intraarticular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal).

[0147] The pharmaceutical compositions and the antibody construct of this invention are particularly useful for parenteral administration, e.g., subcutaneous or intravenous delivery, for example by injection such as bolus injection, or by infusion such as continuous infusion. Pharmaceutical compositions may be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196; 4,439,196; 4,447,224; 4,447, 233; 4,486,194; 4,487,603; 4,596,556;4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163. As described elsewhere herein, the pharmaceutical composition according to the invention is preferably administered intravenously.

[0148] In particular, the present invention provides for an uninterrupted administration of the suitable composition. As a non-limiting example, 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. When exchanging the cartridge in such a pump system, a temporary interruption of the otherwise uninterrupted flow of therapeutic agent into the body of the patient may ensue. In such a case, 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.

[0149] If the pharmaceutical composition has been lyophilized, 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.

[0150] The compositions of the present invention can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, therapeutically effective dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

[0151] 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. For treating tumorous diseases, 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.

[0152] 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. in the form of a leaflet or instruction manual), means for administering the antibody construct of the present invention such as a syringe, pump, infuser or the like, means for reconstituting the antibody construct of the invention and / or means for diluting the antibody construct of the invention. The invention also provides kits for a single-dose administration unit. The kit of the invention may also contain a first recipient comprising a dried / lyophilized antibody construct and a second recipient comprising an aqueous formulation. In certain embodiments of this invention, kits containing single-chambered and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) 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.

[0153] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and 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.

[0154] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0155] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0156] 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.

[0157] 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.

[0158] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having".

[0159] When used herein "consisting of' excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0160] In each instance herein, any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. For example, the disclosure of the term “comprising” includes the disclosure of the terms “consisting essentially of’ as well as the disclosure of the term “consisting of’.

[0161] A better understanding of the present invention and of its advantages will be obtained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.Examples

[0162] Example 1: Effect of an CD16 / EGFR specific Innate Cell Engager on tumor immunology

[0163] 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).

[0164] A) Analysis of pro-inflammatory cytokines

[0165] 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 thebloodstream. 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.

[0166] Levels of IFNy, TNFa and IL10 levels measured at the weekly pre-dose timepoints (C1D8, C1D15, C1D22, C2D1) increased modestly from baseline until C2D1, especially in higher doses of AFM24. However, this increase is in the range of low pg / mL levels, suggesting a cause not directly related to the activation of cells in peripheral blood, but rather at distant healthy or tumor tissue.

[0167] 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.

[0168] Pre-clinical data have shown that AFM24 induces the acute increase of pro- inflammatory cytokines (Wingert S et al., MAbs 2021;13(l): 1950264). In this first-in-human study, IFNg, TNFa, IL6, IL 10, and IL 15 increased after the first dosing, and its values fell back to almost baseline value within 48 h. This is in line with the observations in the nonhuman primates (NHP) (Wingert S et al.).

[0169] IL2 concentrations were at the LLOQ for most of the patients across all time points measured.

[0170] With increasing dose levels, the dose-associated increase of cytokines is less prominent after the first dosing, suggesting less acute release of pro-inflammatory cytokines. An increase of >10 pg / mL IL6 does not occur in patients from doses of 80 mg and above and from the second dosing onwards.

[0171] Levels of IFNY, TNFa and IL 10 levels measured at the weekly pre-dose timepoints (C1D8, C1D15, C1D22, C2D1) increased modestly from baseline until C2D1, especially in higher doses of AFM24. However, this increase is in the range of low pg / mL levels, suggesting a cause not directly related to the activation of cells in peripheral blood, but rather at distant healthy or tumor tissue. (Figure 1, parts A-C)

[0172] B) Immunophenotyping

[0173] 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. As macrophages are mainly based in tissue, in peripheral blood only absolute NK cell numbers were assessed. In this study, 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.

[0175] As there was no clear dose dependency for most analyzed parameters and the overall dataset was rather small, it was decided to group the data into high and low dose, based on the PK parameters. As described in the CSR, a dose-proportional increase in PK from 160 mg onwards was observed indicating saturation of target mediated drug disposition, leading to an increase in half life and steady-state AFM24 levels after 22 to 28 days. The low dose group contains patient data from the patients exposed tol4 mg, 40 mg and 80 mg, while the high dose group contains the data from the patients receivingl60 mg, 320 mg, 480 mg and 720 mg.

[0176] Most of the patients showed a reduction of the relative numbers of peripheral NK cells after the first dosing, which recovered over time (Figure 2). Possible reasons could be cell death or an extravasation of these cells, and distribution to healthy or tumor tissue. A significant increase of cell death is unlikely, as NK cell counts also increase in tumor tissue, however, a contribution of fratricide (NK death mediated by NK cells) cannot not completely be excluded. Relative numbers of monocytes decrease with each. A possible explanation which needs to be confirmed can be an activation, extravasation and differentiation into macrophages.

[0177] Most patients also showed a relative decrease of CD16A levels on NK cells (Figure 2 see graph C). 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. Nevertheless, it cannot be excluded, that CD16Ahigh NK cells partially extravasate after activation and binding to AFM24, thus leading to a relative reduction of CD 16 A.

[0178] 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.). Itwas 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.

[0179] C) Analysis of tumor biopsies

[0180] Biopsies were taken at screening and at day 24 of treatment, although for operational reasons, deviations of several days were accepted.

[0181] C .1 Immunohi stochemi stry

[0182] 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.

[0183] Tumor content ranged from 0% (in two cases) to almost 75%. Biopsies containing no tumor tissue were excluded from further analyses.

[0184] 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).

[0185] Tumor samples were analyzed for the expression of EGFR, CD3, CD8, CD45, CD56, CD68, and CD 163 by IHC. CD3 positive (CD3+) cell density (cell per mm3) was determined by image analysis using the Visiopharm™ software. Except for the clinically validated EGFR assessment, the analyses were not validated and exploratory.

[0186] 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.

[0187] EGFR staining was quantified using the H-Score, which is calculated using low, medium and high intensity staining and the respective positive area of interest.

[0188] EGFR staining at baseline spans from low to high H score. Although variability was high in the obtained H-Score, overall, the EGFR expression by H-score seems not to be altered by AFM24 treatment when comparing baseline and C1D24 results (see Figure 3, parts A and B).

[0189] While the overall number of macrophages (as stained with anti-CD68) was not strongly affected by AFM24 treatment, M2 macrophages (as stained with CD 163) increased in the biopsies of most patients (see Figure 4, parts A and B).

[0190] 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).

[0191] C.2 Expression profiling

[0192] Biopsies from patients who received the higher doses of AFM24 (>160 mg) had additional gene expression profiling (C1D24 versus screening). The assay was not validated and exploratory. 250 ng of total RNA quantified using the NanoDrop™ 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. Finally, the 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.

[0193] 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.

[0194] 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.

[0195] Findings from IHC could be confirmed in the expression profiling, indicating an increase in T cell profiles (Figure 6, parts A and B). Furthermore, an increase in expression of NK cell associated genes could be detected, indicating NK cell engagement and being in line with the AFM24 MoA (Figure 6, part A).

[0196] An increase in NK cell, T cell and cytotoxicity associated functional genes, such as Granzyme, could be shown, suggesting an increase in these cell functions upon AFM24 treatment (Figure 6, part B). For further information on analyzed genes, please see nCounter® PanCancer Immune Profiling Panel.

[0197] D General analysis definitions

[0198] All analyses were performed using SAS® Version 9.4 or later version and R software Version 4.03 or later version. Some analyses were already pre-specified in the AFM24-101 Clinical Trial Protocol and Statistical Analysis Plan and will be reported in the Clinical Study Report (CSR). Therefore, some outputs are only referenced to the CSR and not repeated within this Biomarker Report.

[0199] Example 2: Case studies of patients treated with AFM24-102

[0200] 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.

[0201] 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.

[0202] 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.

[0203] In the ongoing Phase l / 2a clinical trial AFM24-102, the combination of AFM24 with Atezolizumab is investigated in several EGFR expressing tumor types (Non-small cell lung cancer (EGFR- wt), Advanced or metastatic NSCLC harboring a targetable EGFR kinase domain mutation, Gastric / GEJ cancer and Pancreatic / hepatocellular / biliary tract cancer). Initial activity signals have been observed in the NSCLC EGFRwt cohort (Figure 8). Of the 15 initially evaluable patients, four have achieved a confirmed response (one complete response, three partial responses) using RECIST 1.1 criteria and seven patients experienced stable disease. Importantly, seven out of these 15 initially evaluable patients benefited fromthe study treatment, furthermore, all the patients in this cohort have been previously treated with a CPI and have had disease progression on or after these treatments. This is of significant relevance given these patients have very few treatment options available.

[0204] 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.

[0205] The combination of 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. Of note, 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.

[0206] Patients receive 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.Thereafter the 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.

[0207] 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.

[0208] 2.1 Case study 1

[0209] A 74-year-old Asian male Diagnosed in Jun 2020 with Metastatic lung squamous cell carcinoma in Stage IV with one target lesion: Right Upper Lobe (RUL) and two non-target lesions mediastinal lymph nodes and left adrenal gland.

[0210] Treatment history:

[0211] Jul 2020 - Sep 2020: Tiragolumab / placebo + Atezolizumab (CPI mAb targeting PD- 1)BOR = SD Discontinued due to PD

[0212] Oct 2020 - Jun 2022: Cisplatin + pemetrexed + gemcitabineBOR = PR Discontinued due to Toxicity

[0213] May 2023 - Jun 2023: SRF388, an IL-27 targeting antibodyBOR = PD

[0214] For this patient a PR was observed demonstrated by a 50% overall shrinkage of their target lesions with AFM24 and Atezolizumab; details shown in Figure 9.

[0215] 2.2 Case study 2

[0216] A 66-y ear-old male diagnosed in 2019 as 3B, treated with consolidative thoracic radiation therapy (CTRT). Metastatic lung adenocarcinoma TP53 mutated in Stage IV with two target lesions: Left Upper Lobe (LUL) and Left Adrenal gland. No non-target lesions.

[0217] Treatment history:

[0218] Jun 2020 - Mar 2021 : Carboplatin + Pemetrexed BOR = SD Discontinued due toPD

[0219] Mar 2021- Sep 2022 Nivolumab (CPI mAb targeting PD-1)BOR = UK Discontinued due to PD

[0220] Dec 2022- Mar 2023 Docetaxel BOR = SD Discontinued due to PD

[0221] For this patient a PR was observed demonstrated by a 35% overall shrinkage of their target lesions with AFM24 and Atezolizumab; details shown in Figure 10.

[0222] 2.3 Case study 3

[0223] 52-year-old female diagnosed in April 2022 as stage IV. Metastatic lung adenocarcinoma PD-LU in Stage IV with one target lesion: Left Para-aortic lymph node, and three non-target lesions: subcarinal and retroperitoneal lymph nodes; and right lower lobe septal thickening of the lung.

[0224] Treatment history:

[0225] Apr 2022 - Oct 2022 Carboplatin + Pemetrexed + Pembrolizumab (CPI mAb targeting PD-1)BOR = PR Discontinued due to PD

[0226] Nov 2022 - Dec 2022 GEN1046 (Bispecific Antibody Targeting PD-L1 and 4-1BB) - Pembrolizumab (CPI mAb targeting PD-1)BOR = UK Discontinued due to toxicity

[0227] For this patient an ongoing CR was observed with AFM24 and Atezolizumab; details shown in Figure 11.

[0228] 2.4 Case study 4

[0229] 66-year-old male diagnosed in April 2022 as stage IIIB. Locally advanced adenocarcinoma PD-L1 UK, TMB high (26.1 mut / Mb). Stage IIIB with one target lesion: Lung, Left Upper Lobe (LUL), and one non-target lesion: Lymph nodes (subaortic area and left hilar)

[0230] Treatment history:

[0231] Apr 2022 - May 2022 Taxol + Carboplatin BOR = NEDiscontinued due to treatment scheme completion

[0232] Jul 2022 - Jun 2023 Durvalumab (CPI mAb targeting PD-L1) BOR = SD Discontinued due to PD

[0233] The patient received CTRT, and there was evidence of progression in the LUL, therefore qualifying it as a TL before entering the study. Initial response to treatment: Stable Disease. After 4 cycles of treatment: Unconfirmed Partial Response, confirmed after 6 cycles; details shown in Figure 12.

[0234] 2.5 Case study 5

[0235] A 50-year-old female with gastric adenocarcinoma. Diffuse type, signet ring histology Microsatellite stable (MSS), HER2-negative, PD-L1 not evaluated TP53 (V172F) mutation. Stage IV (cutaneous, subcutaneous, bone and peritoneal metastases) with EGFR+ H- score: 190. Diagnosed October 2020.

[0236] Treatment history:

[0237] Nov 2020 - Mar 2021 Oxaliplatin + Capecitabine + Pembrolizumab (CPI mAb targeting PD-1)BOR = PR Discontinued due to PD

[0238] Apr 2021 - Jul 2021 Paclitaxel Discontinued due to PD

[0239] Aug 2021 - Dec 2021 Irinotecan Discontinued due to PD

[0240] Dec 2021 - Jan 2022 FOXP3 inhibitor Discontinued due to PD

[0241] A Partial Response was observed in a gastric cancer patient who had previously progressed on 4 lines of therapy, including anti -PD-1 / chemotherapy combo. While the patient’s skin metastases did not respond to any prior treatment there was a response observed for the treatment with AFM24 in combination with atezolizumab (Figure 13). A CT scan in September 2022 confirmed a 69.01% change in target lesions — PR. CT scan in October 2022 confirmed 70.2% change in target lesions, no change in NTLs, but the patient had new skin lesions; therefore, the patient progressed with a PFS of 8 months and subsequently passed away.

[0242] 2.6 Case study 6

[0243] A 63 -year-old male diagnosed in Jun 2020 as stage IV. Signet Ring Cell metastatic Gastric cancer PD-L1 UK, TP 53 mut. Stage IV with four target lesions: Liver S. Ill, SVIII, LN Para-aortic inferior, LN celiac area, and three non-target lesions: Liver mets, Gastric wall thickening, LN - retroperitoneal.

[0244] Treatment history:

[0245] Jul 2020 - Nov 2021 Carboplatin - Etoposide - Nivolumab (CPI mAb targeting PD- 1)BOR = PR Discontinued due to PD

[0246] Dec 2021- Jan 2022 Carboplatin - EtoposideBOR = PD Discontinued due to PD

[0247] Feb 2022 - Aug 2022 Capecitabine - OxaliplatinBOR = SD Discontinued due to PD

[0248] Aug 2022 - Jan 2023 Folinic Acid - 5FU - IrinotecanBOR = SD Discontinued due to PD

[0249] A Partial Response was observed for this patient in 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; details shown in Figure 14.

[0250] 2.7 Case study 7

[0251] 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. Stage IV with four target lesions: Liver cupula, liver segment VII, LN - celiac, LN - retroperitoneal, and three non-target lesions: Liver, LN - infradiaphragmatic, peritoneal carcinomatosis.

[0252] Treatment history:

[0253] Apr 2022 - Nov 2022 Folinic Acid - 5FU - OxaliplatinBOR = PR Discontinued due to PD

[0254] Jan 2023 - Feb 2023 IrinotecanBOR = NE Discontinued due to toxicity

[0255] Feb 2023 - Apr 2023 PaclitaxelBOR = NE Discontinued due to PD

[0256] A Partial Response was observed for this patient in the present study. Patient was on SD until TA3 (-26.3% compared to baseline). Change in SOD compared to baseline: - 32% Non-Target lesions were evaluated as present (without changes); details shown in Figure 15.

[0257] Abbreviations:

[0258] BOR: Best Observed Response

[0259] CR: Complete Response

[0260] NE: Not Evaluable

[0261] PD: Progressive Disease

[0262] PR: Partial Response

[0263] SD: Stable Disease

[0264] SOD: Sum Of Diameters

[0265] UK: Unknown

[0266] Example 3: Similarity of effects observed for different polyspecific CD16A / solid tumor antigen binder

[0267] AFM24 is a bispecific CD16A / EGFR specific antibody construct which enables engagement of CD16A+innate effector cells with EGFR+target cells. As described herein above EGFR is a solid tumor associated target antigen. AFM32 is a bispecific CD16A / FOLR1 specific antibody construct which enables engagement of CD16A+innate effector cells with FOLR1+target cells. As described herein above FOLR1 is a solid tumor associated target antigen. Thus, both antibody constructs are innate cell engagers (ICE), which enable the engagement of innate effector cells with solid tumor specific target cells.

[0268] As shown in the following experiments, the effects of the solid tumor specific ICE molecules AFM24 and AFM32 show similar characteristics.

[0269] A) Antibody-induced release of IL-6, TNF-a, and IFN-y in human PBMC cultures in presence target cells

[0270] 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)

[0271] 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.

[0272] An AFM24-mediated release of TNF-a was consistently detected in all analyzed PBMC samples after 4h incubation with potency values ranging from 5.6pM to 642.6pM and a mean maximal efficacy of 187.3pg / mL. These data suggest that the high affinity interaction of AFM24 with CD16A on NK cells and with EGFR on target cells is required to stimulate IFN-y release, in contrast to the low-affinity Fc-based interaction of cetuximab which had only a marginal effect on IFN-y induction. The release of the cytokines IL-6, TNF-a and IFN- y was 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).

[0273] The data demonstrate that AFM24-induced the release of IL-6, TNF-a, and IFN-y, but not of IL-2, IL-4, and IL- 10 in cultures of PBMC from healthy donors in the presence of EGFR+A-431 target cells. The release was AFM24 concentration-dependent and occurred exclusively in the presence of EGFR-expressing target cells. After 24h, AFM24 induced release of IL-6 with a maximal efficacy of up to 1569.8pg / mL and a mean potency of 22.5pM. Highest levels of TNF-a and IFN-y were detected after 4h stimulation with AFM24 resulting in a mean Emax of 187.3pg / mL for TNF-a and 17.2pg / mL for IFN-y release. The AFM24 stimulated release of IL-6, TNF-a, and IFN-y was confirmed in a comprehensive study using 10 PBMC donor preparations and four different AFM24 drug substance batches, demonstrating reproducible release of the three cytokines despite donor-to-donor variability and inter-assay variation. The absence of release of all tested cytokines upon stimulation with the control antibodies scFv-IgAb_44 (anti-RSV / CD16A) and scFv-IgAb_45 (anti- EGFR / RSV) demonstrates the necessity of bispecific engagement and crosslinking of effector and target cells for mediating cytokine release in vitro. In comparison, the anti -EGFR IgGl, cetuximab, induced the release of IL-6 and TNF-a, but not of IFN-y. This difference may be explained by the specific and high affinity interaction of AFM24 with CD16A compared to the low affinity interaction of the Fc-part of cetuximab with Fey receptors.

[0274] Depletion of specific PBMC subsets revealed that in the absence of CD14+or CD33+mononuclear cells the release of IL-6 and TNF-a was substantially reduced, demonstrating that monocytes are required for the secretion of the two cytokines. Since monocytes are described to produce IL-6 and TNF-a, it is tempting to speculate that AFM24 stimulates monocytes to produce the two cytokines in the presence of target cells. IFN-y secretion was strongly decreased in PBMC cultures upon NK cell depletion, suggesting that NK cells are the main cell population responsible for IFN-y release.

[0275] Notably, 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.

[0276] 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). The addition of FRa-positive OVCAR-3 cells to PBMC slightly stimulated antibody-independent release after 4 h incubation. Upon 4 h co-culture of PBMC with FRa-positive OVCAR-3 cells, 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.

[0278] 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. Upon co-culture of PBMC with FR a -positive OVCAR-3 cells 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.

[0279] In the absence of target cells neither AFM32 nor the control items induced IFN-y release after 4 h or 24 h. Upon co-culture of PBMC with FR a -positive OVCAR-3 cells mean IFN-y release was strongly induced by AFM32 with one donor exhibiting a more pronounced release than the other two donors. In addition, human IgGl anti-FR a IgAb_335 and scFv- IgAb_162 (RSV / CD16A) also enhanced IFN-y release in the presence of target cells after 4 h and 24 h incubation. The effect was more pronounced after 24 h (mean: 7 pg / mL for IgAb_335 and 10 pg / mL for scFv-IgAb_162 FOLR1 / RSV scFv-IgAb compared to 2 pg / mL without antibody).

[0280] The data show that AFM32 does not induce the release of substantial amounts of pro- inflammatory cytokines in cultures of human PBMC in the absence of FRa-positive target cells. 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 (RSV / CD16A) 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. In contrast, 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.

[0281] These data support the specificity of AFM32-induced immune cell activation and cytokine release, and the requirement of both, binding to CD16A-positive immune cells and to FRa-positive target cells for efficacious immune cell activation by AFM32.

[0282] These data demonstrate that engagement of PBMC by AFM32 in the presence of FRa- expressing target cells is associated with the release of pro-inflammatory cytokines such as IL-6, IFN-y, and TNF-a, however no substantial cytokine-release is measured in the absence of target cells.

[0283] B) AFM24 and AFM32 induced antibody-dependent cellular phagocytosis (ADCP) of target positive solid tumor cells by macrophages

[0284] 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. Depending on the tumor indication, 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. Antibody-dependent cellular phagocytosis (ADCP) was measured by flow cytometry using in vitro differentiated macrophages derived from healthy donor PBMC, along with two tumor cell lines expressing different levels ofEGFR or FOLR1 and one EGFR or FOLR1 negative cell line as target cells. Details of such ADCP assay are described e.g. in Wingert et al., Mabs 2021, VOL. 13, NO. 1.

[0285] 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. In contrast, 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. Together, these data demonstrate an efficient and EGFR-specific AFM24-induced ADCP that appears to be unrelated to the EGFR expression level or KRAS mutational status of the tumor target cell, see Figure 18.

[0286] For AFM32 antibody-dependent cellular phagocytosis (ADCP) was measured by flow cytometry using in vitro differentiated macrophages derived from healthy donor CD 14+ monocytes, along with FRa+ tumor cell lines (HeLa and HCC-78) expressing different levels of FRa. Furthermore, it was also investigated whether macrophages would phagocytose NK cells. AFM32 only induced phagocytosis of the FRa+ HeLa and HCC-78 cell lines, while NK cells were unaffected, demonstrating strict antigen-specificity, see Figure 19. Altogether, these data demonstrate an efficient and FRa-specific AFM32-induced ADCP that is most likely mediated by the bivalent and specific binding of AFM32 to FRa on tumor target cells. At the same time, it could be shown that the two anti-CD16A binding domains in the C- terminal scFv of AFM32 do not allow cross-linking of NK cells with macrophages and thus prevent phagocytosis of NK cells.

[0287] C) AFM24 and AFM32 demonstrate similar in vivo anti-tumor control against target positive tumor cells

[0288] The anti-tumor activity of AFM24 was assessed in hIL-15 NOG mice inoculated i.p. with luciferase-transduced EGFR-expressing TNBC MDA-MB-231 tumor cells (5.0 x 105cells / mouse, n=8 per group). On day 2, mice received either vehicle alone or NK cells (2.7 x 106cells / 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.

[0289] All mice had received human IL-2 three times per week.

[0290] BLI, bioluminescence imaging; hIL-15, human interleukin- 15; IL-2, interleukin-2; i.p., intraperitoneally; TNBC, triple negative breast cancer

[0291] AFM24, in combination with adoptive NK cells, leads to dose-dependent tumor regression in a mouse xenograft model, see Figure 20, part B.

[0292] Dose titration of AFM32 in B-hCD16A (CB-17 SCID) mice bearing subcutaneous SKOV3 Red FLuc tumors.

[0293] 7-9 weeks-old female B-hCD16A (CB-17 SCID) mice were subcutaneously injected with 5 x

[0294] 106SKOV3 Red FLuc cells per mouse in 100 pL of 1 : 1 matrigel: serum-free medium mix. Animals were grouped into 5 groups of 8 mice per group 18 days post-inoculation when tumor volumes (TV) reached mean 109.24 mm3(106.66-111.42 mm3) and were treated twice a week with IV injections of either vehicle (Gl) or AFM32 at different doses (G2-G5: 3, 10, 30, 60 mg / kg) until 28 days post-treatment initiation (Day 46). TVs and BWs were measured twice weekly until the last observation day and the animals were euthanized for sample collection. A subset of terminal tumor samples was processed for subsequent flow cytometry analysis.On Day 46 post-inoculation, the TV (mean ± SEM) of animals in Vehicle control (Gl) was 527.29 ± 15.53 mm3(469.44 - 591.30 mm3). Treatment with AFM32 demonstrated significant antitumor efficacy compared to the control at all the tested doses (3, 10, 30, 60 mg / kg) with TGI of 54.21%, 63.10%, 71.78%, 74.69% respectively, resulting in TV (mean ± SEM) of 300.65 ± 6.73 mm3, 263.50 ± 4.94 mm3, 227.22 ± 5.54 mm3, 215.04 ± 3.62 mm3(P<0.0001, Repeated measure (RM) two-way ANOVA with Geisser-Greenhouse correction, Dunnett’s multiple comparisons test). AFM32 treatment also resulted in significant reduction in terminal tumor weights. Tumor weight inhibition (TWI) of 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.Twice-weekly IV doses of AFM32 (3, 10, 30 and 60 mg / kg) demonstrated a significant antitumor response compared to the vehicle control, see Figure 21. In this study, no unexpected clinical observations or deaths or BW losses were noted indicating that the AFM32 treatment up to 60 mg / kg is well-tolerated and safe in female B-hCD16A (CB-17 SCID) mice.Sequence Listing

Claims

Claims1. A method of treating a solid tumor in a subject, and wherein the subject is, or is predicted to be, resistant to immune checkpoint inhibitor cancer treatment, the method comprising 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 administering to the subject an immune checkpoint inhibitor, optionally a PD-L1 or PD-1 inhibitor.

2. The method of claim 1 wherein the solid tumor characterized by epidermal growth factor receptor (EGFR) overexpression on solid tumor cells, and the polyspecific binding molecule binds to EGFR.

3. The method of claim 1 or 2 wherein the solid tumor is selected from the group consisting of lung cancer, colorectal cancer, liver cancer, brain cancer, kidney / renal cancer, ovarian cancer, breast cancer, squamous cell carcinoma, bladder cancer, head and neck cancer, gastric cancer, esophagus cancer, sarcoma, mesothelioma, and adenocarcinoma, optionally, non-small cell cancer (NSCLC), hepatocellular carcinoma (HCC), glioblastoma (GBM), Clear Cell Renal Cell Carcinoma (ccRCC), Triple-negative breast cancer (TNBC), squamous cervical cancer, and squamous cell carcinoma of head and neck (SCCHN).

4. The method of any of the previous claims wherein the solid tumor is lung cancer, optionally non-small cell cancer (NSCLC) or mesothelioma.

5. The method of any of claims 1-3 wherein the solid tumor is adenocarcinoma or squamous cell carcinoma.

6. The method of claim 1 wherein the solid tumor is characterized by Folate Receptor 1 (FOLR1) FOLR1 overexpression on solid tumor cells, and the polyspecific binding molecule binds to FOLR1.

7. The method of claim 6, wherein said solid tumor is selected from the group consisting of ovarian cancer, breast cancer, lung cancer, colorectal cancer, renal cancer, pancreatic cancer, endometrial cancer, and brain cancer, optionally, high grade serous ovarian cancer orepithelial ovarian cancer, TNBC, NSCLC, mesotheliomas, ccRCC, Pancreatic ductal adenocarcinoma (PDAC), and non-malignant endometrial cancer.

8. The method of any of the previous claims wherein the polyspecific binding molecule is a bispecific binding molecule.

9. The method of any of the previous claims wherein the poly specific binding molecule (a) does not elicit EGFR or FOLR1 downstream signalling, (b) the EGFR-expressing or FOLR1- expressing solid tumors do not have driver or resistance mutation(s), or both (a) and (b).

10. The method of any of the previous claims wherein the subject is resistant to immune checkpoint inhibitor cancer treatment.

11. The method of claim 10 wherein the immune checkpoint inhibitor that the subject is resistant to is selected from ipilimumab, pembrolizumab, nivolumab, cemiplimab, dostarlimab, atezolizumab, durvalumab, and avelumab.

12. The method of any of the previous claims wherein the subject has undergone prior treatment for the solid tumor.

13. The method of claim 12 wherein the prior treatment for the solid tumor included (a) a platinum -based therapeutic treatment, (b) a PD-1 or PD-L1 inhibitor treatment, or both (a) and (b).

14. The method of claim 12 wherein the subject has undergone prior treatment with two or more different treatment regimens which were unsuccessful and / or discontinued.

15. The method of claim 12 wherein the subject has an actionable driver mutation other than an EGFR mutation, and has undergone prior treatment for the mutation.

16. The method of claim 15 wherein 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.

17. The method of claim 2, wherein the polyspecific binding molecule is administered to the subject once every about 6 to about 8 days, or once every about 7 days.

18. The method of claim 2 or 17, wherein the polyspecific binding molecule is administered at a dose in the range of 7-8 mg / kg.

19. The method of any one of claims 2, 17, and 18, wherein the PD-L1 inhibitor is administered to the subject once every about 13 to about 15 days, or once every about 14 days.

20. The method of any one of claims 2 and 17-19, wherein the PD-L1 inhibitor is administered at a dose in the range of 14-16 mg / kg.

21. The method of any of the previous claims, wherein the poly specific binding molecule and the PD-L1 inhibitor are administered to the subject at the same time, or about the same time.

22. The method of claim 21, wherein 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.

23. The method of any of the previous claims, wherein 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.

24. The method of any of the previous claims, wherein a cycle of administering consists of administering the polyspecific binding molecule 8 times, and administering the PD-L1 inhibitor 4 times, and treatment of the subject is carried out for at least one cycle.

25. The method of any of the previous claims, wherein the poly specific binding molecule and the PD-L1 inhibitor are administered intravenously.

26. The method of any of the previous claims, comprising assessing tumor regression after each cycle of treatment.

27. The method of any of the previous claims, wherein treating results in about 5% (size) or greater shrinkage of tumor, about 10% or greater shrinkage of tumor, about 20% or greater shrinkage of tumor, about 30% or greater shrinkage of tumor, or about 50% or greater shrinkage of tumor.

28. The method of any of the previous claims wherein treating causes cytotoxic T cells to migrate into the tumor and cause a reduction in tumor mass.

29. A composition for treating a solid tumor in a subject 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.

30. The composition of claim 30, wherein the tumor antigen is EGFR.

31. The composition of claim 30, wherein the tumor antigen is FOLR1.

32. The composition of any of claims 29-31, wherein the PD-L1 inhibitor is present in an amount that is in the range of 1.25 - 1.75 times greater than an amount of polyspecific binding molecule.

33. The composition of any of claims 29-32, in liquid form and configured for IV administration.

34. The composition of any of claims 29-33, comprising one or more excipients.

35. A method of re-sensitizing a subject to immune checkpoint inhibitor cancer treatment, the method comprising administering to a subject who is, or is predicted to be, resistant to immune checkpoint inhibitor cancer treatment a poly specific binding molecule that, at least, binds CD16A on innate immune cells and a tumor antigen on tumor cells.

36. The method of claim 35, wherein the subject is a solid tumor patient.

37. A method of transforming a cold tumor into a hot tumor in a patient, the method comprising administering to a subject having a cold tumor, and who optionally is, or optionally 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.

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