Anthracycline-containing binding protein-toxin fusions and their use in immuno-oncology applications - Patents.com

Abstract

The present invention relates to binding protein-toxin fusions containing one or more anthracycline toxin moieties and their use in immuno-oncology applications.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to binding protein-toxin fusions containing one or more anthracycline toxin moieties, and their use in immuno-oncology applications.

[0002] 2. Background of the Invention Binding protein-toxin fusions, of which antibody-drug-fusions (ADCs) are the most prominent representatives, have had an enormous impact in cancer therapy, providing new therapeutic approaches to previously untreatable tumor types.

[0003] Meanwhile, the discovery of immune checkpoints and the development of immune checkpoint inhibitors have brought renewed focus on the role of the immune system in cancer treatment and how to stimulate the immune system or overcome the immune checkpoints that each tumour uses to defend itself against immune system attack.

[0004] Although both approaches have been much debated and have represented significant advances in the treatment of cancer, it has become apparent that there are tumor types that cannot be treated with these new approaches.

[0005] For example, ADCs bearing certain toxins have been shown to be inactive against certain cancer types, even though they address cancer-specific antigens with their antibodies, and there are tumor types for which immune checkpoint inhibitors are ineffective.

[0006] Therefore, one object of the present invention is to provide a new approach to treat tumor types that have not previously been addressed by existing therapies.

[0007] A further object of the present invention is to provide combination therapies that increase the antitumor activity of the corresponding monotherapies.

[0008] The invention and further objects are achieved by the methods and means set forth in the independent claims. The dependent claims relate to particular embodiments.

[0009] Summary of the Invention The present invention provides binding protein-toxin fusions comprising one or more anthracycline toxin moieties, and their use in immuno-oncology applications. The general advantages of the invention and its features are explained in detail below. [Brief description of the drawings]

[0010] [Figure 1] 1 is a chemical structure of the pentaglycine-modified EDA-anthracycline PNU-159682 derivative (G5-EDA-PNU or G5-PNU) used to generate site-specifically fused ADCs used in the experiments of the present invention. G3-PNU and G2-PNU refer to the same structure, except that pentaglycine is replaced by triglycine or diglycine, respectively.

[0011] [Diagram 2] Figure 2 is a FACS analysis of the expression of human ROR1 stably expressed on mouse EMT-6 breast cancer cells. EMT-6-ROR1 clone 14, selected for in vivo studies, was analyzed by FACS staining for ROR1 expression using fluorescently labeled anti-ROR1 antibody clone 2A2. The negative control shows staining of the same cells with a fluorescently labeled isotype-matched control antibody.

[0012] [Diagram 3] Figure 3 shows dose escalation of the novel anti-ROR1 PNU-ADC based on antibody XBR1-402 with single treatments at different dose levels (0.25, 0.5 and 1 mg / kg) in the orthotopic syngeneic breast cancer model EMT-6-ROR1 (clone 14). (A) Tumor growth of orthotopically implanted breast tumors is monitored over time in the different treatment groups as indicated. (B) Tumor growth curves in individual mice of the groups shown in panel (A) are shown.

[0013] [Figure 4] Figure 4 shows the change in tumor volume in syngeneic mice orthotopically implanted with EMT-6-ROR1 (clone 14) breast cancer cells. (A) Injection of vehicle control (PBS), or (B) single dose of isotype-matched control PNU-ADC containing anti-CD30 antibody brentuximab, clone Ac10, at 0.25 mg / kg, or (C) single dose of anti-ROR1 PNU-ADC containing novel anti-ROR1 antibody XBR1-402 at 0.25 mg / kg, which according to Figure 3 has been determined to be a suboptimal dose for the treatment of orthotopic EMT-6-ROR1 breast tumors in mice. Panels (D) and (E) show combination treatment with anti-CTLA4 immune checkpoint inhibitor antibody 9D9 with isotype-matched control ADC Ac10 at 0.25 mg / kg and novel anti-ROR1 PNU ADC at 0.25 mg / kg, as indicated. (F) shows the median group tumor volume evolution (Last-Observation-Carried-Forward (LOCF) method was applied) for the following groups: vehicle control, Ac10-G5-PNU isotype control, and XBR1-402-G5-PNU; (G) shows the median group tumor volume evolution (Last-Observation-Carried-Forward (LOCF) method was applied) for the following groups: vehicle control, Ac10-G5-PNU isotype control, and anti-mouse CTLA-4 antibody 9D9, and XBR1-402-G5-PNU, and anti-mouse CTLA-4 antibody 9D9;

[0014] [Diagram 5]Figure 5 shows FACS analysis of expression of stably expressed human ROR1 in: (A) mouse CT-26 breast cancer cells (clone 3) and (B) B16 mouse melanoma cancer cells (pool). CT-26-ROR1 clone 3 and B16-ROR1 pool, selected for in vivo studies, were analyzed by FACS staining for ROR1 expression with fluorescently labeled anti-ROR1 antibody clone XBR1-402 (right peak). Negative control shows staining of the same cells with fluorescently labeled isotype-matched control antibody (left peak).

[0015] [Figure 6] FIG. 6 shows tumor volume evolution in mice implanted with CT-26-ROR1 clone 3 tumor cells according to Example 6. (A) Vehicle control (untreated) in group 1, (B) Isotype control in group 2, (C) Anti-ROR1 ADC huXBR1-402-17 in group 3, (D) Isotype control + immune checkpoint inhibitor anti-mouse PD-1 antibody in group 4, (E) Anti-ROR1 ADC huXBR1-402-17 + immune checkpoint inhibitor anti-mouse PD-1 antibody in group 7. The insets in panels A-E provide the number of mice in which tumor growth was inhibited compared to the number of mice in each treatment group. The results suggest cooperation between the evaluated immune checkpoint inhibitors and the targeted PNU derivative-containing ADC.

[0016] [Figure 7]FIG. 7 shows lung surface colony volumes in mice after intravenous application of the B16 pool overexpressing ROR1, followed by treatment with vehicle control ("Vehicle", group 1), immune checkpoint inhibitor anti-mouse CTLA-4 antibody ("CTLA-4", group 2), anti-ROR1 ADC huXBR1-402-17 ("ADC", group 3), and anti-ROR1 ADC huXBR1-402-17 + immune checkpoint inhibitor anti-mouse CTLA-4 antibody ("ADC + CTLA-4", group 4), as in Example 7. These results suggest a strong synergy between the evaluated immune checkpoint inhibitors and ADCs, including targeted PNU derivatives, in poorly immunogenic tumors. "ns" indicates not statistically significantly different, and "*" indicates statistically significantly different.

[0017] [Figure 8] Figure 8 shows CD4 and CD8 staining of hROR1 (human ROR1)-overexpressing B16 tumor cryosections from (A) untreated control mice, and (B) three mice treated with ADCs according to the present invention (one dose at 4 mg / kg). As shown in Figure 8(A), B16 tumors naturally contain low levels of CD4+ and CD8+ cells. Figure 8(B) shows that treatment with ADCs increases the number of CD4+ and CD8+ cells in the tumor, as shown in the present invention.

[0018] [Figure 9] Figure 9 shows the analysis of 41BB (CD137) staining, a T cell activation marker, on infiltrating lymphocytes in hROR1 (human ROR1) overexpressing CT-26 and B-16 tumors. As shown in Figure 9, the number of activated T CD4+ (A) and CD8+ (B) lymphocytes is significantly lower in B-16 tumors when compared to CT-26 tumors when B16 tumors are maintained as cold tumors. "*" indicates a statistically significant difference.

[0019] Detailed Description of the Invention Before describing the present invention in detail, it is to be understood that the invention is not limited to the particular component parts of the devices or process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural references unless the context clearly dictates otherwise. Additionally, when a parameter range delimited by numerical values ​​is given, it should also be understood that the range is deemed to be inclusive of those limits.

[0020] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be construed as separate embodiments unrelated to each other. Features discussed in the context of one embodiment are intended to be disclosed in conjunction with the other embodiments presented herein. In one instance, if a particular feature is not disclosed in one embodiment and is disclosed in another embodiment, one skilled in the art will understand that this does not necessarily mean that the feature is not disclosed in the other embodiment. Those skilled in the art will appreciate that while it would be a purpose of this application to disclose the above features for other embodiments, this has not been done for the sake of clarity and to keep the specification to a manageable volume.

[0021] Furthermore, the contents of the prior art documents described herein are incorporated by reference. This refers in particular to prior art documents that disclose standard or routine methods. In that case, incorporation by reference has the primary purpose of providing a full enabling disclosure and avoiding lengthy repetition.

[0022] According to one aspect of the invention, there is provided a binding protein-toxin fusion comprising one or more anthracycline toxin moieties fused to a binding protein for (the manufacture of a medicament for) the treatment of a patient: Patients suffering from a neoplastic disease characterized as a cold tumor, Patients at risk of developing a neoplastic disease characterized as a cold tumor, and / or Patients who have been diagnosed with a neoplastic disease characterized as a cold tumor.

[0023] It is important to highlight that the above terminology in brackets, “for the manufacture of medicinal products”, has been selected to meet different national / regional requirements for claims directed to the medical use of a given compound. This term should in particular encompass both the so-called Swiss-type format (where the modifier "for the manufacture of medicinal products" is required) and the respective claim language under EPC 2000 (where the same modifier is no longer permitted).

[0024] According to another aspect of the invention, there is provided a method for treating or preventing a neoplastic disease characterized as a cold tumor, said method comprising administering to a patient a binding protein-toxin fusion comprising one or more anthracycline toxin moieties.

[0025] According to another aspect of the invention, the combination of: (i) a binding protein-toxin fusion comprising one or more anthracycline toxin moieties fused to a binding protein; and (ii) immune checkpoint inhibitors; Provided for the treatment of patients (for the manufacture of medicines) Patients suffering from a neoplastic disease characterized as a cold tumor, Patients at risk of developing a neoplastic disease characterized as a cold tumor, and / or Patients who have been diagnosed with a neoplastic disease characterized as a cold tumor.

[0026] Here, the binding protein-toxin fusion and the immune checkpoint inhibitor are administered to the patient simultaneously or sequentially in any order.

[0027] According to another aspect of the present invention there is provided a method of treating or preventing a neoplastic disease characterized as a cold tumor, said method comprising administering to a patient: (i) a binding protein-toxin fusion comprising one or more anthracycline toxin moieties fused to a binding protein; and (ii) Immune checkpoint inhibitors.

[0028] Here, the binding protein-toxin fusion and the immune checkpoint inhibitor are administered to the patient simultaneously or sequentially, in any order.

[0029] According to another aspect of the invention, a binding protein-toxin fusion comprising one or more anthracycline toxin moieties attached to a binding protein is provided for use in combination with an immune checkpoint inhibitor (for the manufacture of a medicament for) use in the treatment of a patient: Patients suffering from a neoplastic disease characterized as a cold tumor, Patients at risk of developing a neoplastic disease characterized as a cold tumor, and / or Patients who have been diagnosed with a neoplastic disease characterized as a cold tumor.

[0030] According to another aspect of the invention is an immune checkpoint inhibitor for use in combination with a binding protein-toxin fusion comprising one or more anthracycline toxin moieties fused to a binding protein, for use in the treatment of a patient (for the manufacture of a medicament for): Patients suffering from a neoplastic disease characterized as a cold tumor, Patients at risk of developing a neoplastic disease characterized as a cold tumor, and / or Patients who have been diagnosed with a neoplastic disease characterized as a cold tumor.

[0031] In one embodiment, the immune checkpoint inhibitor modulates a positive immune checkpoint protein, hi another embodiment, the immune checkpoint inhibitor modulates a negative immune checkpoint protein.

[0032] In one embodiment, a binding protein-toxin fusion comprising one or more anthracycline toxins is administered prior to the immune checkpoint inhibitor.

[0033] In one embodiment, the patient is suffers from a neoplastic disease characterized as a cold tumor; and / or Have been diagnosed with a neoplastic disease characterized as a cold tumor.

[0034] A "hot tumor" refers to a cancerous or neoplastic tissue characterized by infiltration of immune cells. The level of immune infiltration reflects whether the immune system recognizes and engages the tumor. The term "hot tumor" is used herein synonymously with the term "immunoresponsive tumor." Alternatively, a "hot tumor" may be defined as a tumor that responds to treatment with an immune checkpoint inhibitor.

[0035] As used herein, the term "responding to treatment" means a statistically significant reduction in tumor burden or growth compared to comparable vehicle or isotype control treatment.

[0036] In one embodiment, the term "cold tumor" defines cancerous or neoplastic tissue characterized by poor or absent immune cell infiltration. The term "cold tumor" is used herein synonymously with the term "immunologically unresponsive tumor."

[0037] One criterion for a tumor to be considered "cold" is the so-called Immunoscore, which is based on the density of two types of T cells in a tumor sample. One example is the density of two lymphocyte populations (CD3 / CD45RO, CD3 / CD8 or CD8 / CD45RO) both in the tumor core (CT) and in the tumor invasive margin (IM). Immunoscore provides scores ranging from Immunoscore 0 (I0), where low densities of both cell types are found in both regions, to Immunoscore 4 (I4), where high densities are found in both regions.

[0038] Methods for determining the Immunoscore are described, for example, in Galon et al., 2014, Pages et al., 2009, Angell et al., 2013, and Galon et al., 2013, the contents of which are incorporated herein by reference.

[0039] It has been argued previously that tumors with low Immunoscores ("cold tumors") respond poorly to treatment with immune checkpoint inhibitors such as anti-PD-1, anti-CTLA-4 or anti-PD-L1 antibodies, whereas tumors with high Immunoscores respond to such treatment (see Bu et al., 2016). Without being bound by theory, the rationale behind this finding may be that if there is no immune involvement in the respective tumor, blocking immune checkpoints with a given inhibitor cannot change anything for the better.

[0040] The present inventors have realised that binding protein-toxin fusions containing one or more anthracycline toxin moieties have particular immunological efficacy in tumors, inter alia providing immune protection from tumor rechallenge. This surprising finding was not anticipated from findings made with immune checkpoint inhibitors, such as anti-PD-1, anti-CTLA-4 or anti-PD-L1 antibodies. The rationale that applies to the latter likely does not apply to binding protein-toxin fusions that contain one or more anthracycline toxin moieties.

[0041] According to one embodiment of the present invention, a neoplastic disease characterized as a cold tumor is a tumor that is unresponsive, resistant to immune checkpoint inhibitor treatment, or that recurs after immune checkpoint inhibitor treatment.

[0042] As used herein, the term "non-responsive" refers to a tumor that does not respond or has not responded reasonably in a favorable manner in terms of tumor shrinkage or duration of stabilization or shrinkage in response to treatment with an immune checkpoint inhibitor.

[0043] As used herein, the term "resistant" refers to tumors that do not or have not responded reasonably in a favorable manner in terms of tumor shrinkage or duration of stabilization or shrinkage in response to treatment with an immune checkpoint inhibitor for three months or more. As used herein, the term "recurrent" refers to a tumor that has returned following treatment with an immune checkpoint inhibitor, usually after a period in which the tumor could not be detected. The tumor may return to the same location or another location in the subject's body.

[0044] Such treatment with immune checkpoint inhibitors was preferably as a single agent.

[0045] Such treatment with immune checkpoint inhibitors is preferably administered at the dose or regimen recommended in the respective drug package insert or SOPC.

[0046] According to one embodiment of the present invention, the neoplastic disease characterized as a cold tumor is selected from the group consisting of: ·melanoma, colon cancer or colon tumors, Pancreatic cancer and tumors Glioblastoma, ovarian cancer or tumors, and / or Prostate cancer or tumors.

[0047] According to one embodiment of the invention, the binding protein binds to at least one target selected from the group consisting of: ROR1 CS1 HER2 Mesothelin (MN) and / or ROR2.

[0048] Among these, ROR1 is a preferred target.

[0049] These targets, and the antibodies they capture, are described in the following publications: US2019112385A1 (Mesothelin) ·US2019153092A1, WO2019016381(ROR1) WO2019030240A1(CS-1), US2018028682 (HER2) and WO2019016392A1(ROR2), The contents of which are incorporated herein by reference.

[0050] Preferably, said target is of human origin. In particular, ROR1, when referred to herein, preferably means human ROR1.

[0051] As used herein, the term "immune checkpoint inhibitor" refers to any binding agent or compound suitable for acting against an immune checkpoint protein. In particular, "immune checkpoint inhibitors" refers to any binding agent or compound suitable for modulating the activity of immune checkpoint proteins to support the activity of the immune system, in particular the ability of T cells to recognize and act against cancer cells. Immune checkpoint proteins can be positive (i.e., they support T cell activity and the immune response) or negative (i.e., they limit T cell activity and the immune response). In the case of a positive immune checkpoint protein, an immune checkpoint inhibitor refers to a binding agent or compound suitable for promoting the activity of this immune checkpoint protein. In the case of a negative immune checkpoint protein, an immune checkpoint inhibitor refers to a binding agent or compound suitable for inhibiting the activity of this immune checkpoint protein.

[0052] Immune checkpoints are proteins that regulate the immune system (Pardoll DM "The blockage of immune checkpoints in cancer immunotherapy", Nature, 2012, vol. 12, pp. 252-264) and are crucial for self-tolerance, which prevents the immune system from attacking cells indiscriminately, and are preferably selected from the group disclosed in the following table: [Table 1] For example, the PD-1 checkpoint protein expressed on the surface of T cells acts to stop those T cells from attacking other cells in the body. When T cell PD-1 binds to PD-L1 on the cell surface, the T cell does not mount an immune response against that cell. Certain cancer cells express high levels of PD-L1, which helps them evade immune system attack. Therefore, antibodies that bind to either PD-1 or PD-L1 and block their binding to each other are useful for promoting an immune response against cancer cells. CTLA-4 is another such negative immune checkpoint protein expressed on T cells.

[0053] According to one group of embodiments, the neoplastic disease is characterized as a cold tumor, characterized by having an Immunoscore of I<1.

[0054] Immunoscore is a method for predicting the prognosis of cancer patients based on the immune cells that infiltrate and surround the cancer. Immunoscore has been internationally validated in colorectal cancer. This builds on the findings of Galon et al. (2006), who demonstrated a positive association between cytotoxic and memory T cells and survival in patients with colorectal cancer.

[0055] Immunoscore incorporates the influence of the host immune response into cancer classification, improving prognostic accuracy. The density of two T lymphocyte populations (CD3 / CD8, CD3 / CD45RO or CD8 / CD45RO) in the center and periphery of the tumor is measured. Immunoscore provides a score ranging from 0 (I0), if low densities of both cell types are found in both regions, to Immunoscore 4 (I4), if high densities are found in both regions. Immunoscore does not measure how immune cells are organized at the periphery of a tumor or the amount of B cells, tertiary lymphoid structures and germinal centers, all of which play important roles in the immune response to cancer, including colorectal cancer.

[0056] In one embodiment, a cold tumor is a tumor with an immunoscore of I0 or I1, preferably an immunoscore of I1.

[0057] In another embodiment, the term "cold tumor" defines a cancerous or neoplastic tissue characterized by absent or insufficient immune cell infiltration. The level of immune infiltration reflects whether the immune system recognizes the tumor.

[0058] In another embodiment, a cold tumor is a tumor that does not respond to an immune checkpoint inhibitor when the immune checkpoint inhibitor is administered in vivo as monotherapy. In this embodiment, compared to an immunocompetent mouse model, a cold tumor is a tumor that, once established in the mouse, does not respond significantly to an immune checkpoint inhibitor administered as monotherapy at up to about 10 mg / kg, or up to 20 mg / kg, or up to the maximum tolerated dose (MTD) in that mouse model (compared to a vehicle control group). In humans, a cold tumor is, in another embodiment, a tumor that does not respond significantly to an immune checkpoint inhibitor administered as monotherapy according to a dosage regimen of the immune checkpoint inhibitor, e.g., up to 10 mg / kg, e.g., 1 mg / kg, 2 mg / kg, 3 mg / kg, or 200 or 240 mg, optionally every 2-3 weeks.

[0059] In another embodiment, cold tumors can be defined in comparison to human solid tumors by comparison to the RECIST (Response Evaluation Criteria In Solid Tumors) criteria published in February 2000 (Therasse P. et al., "New Guidelines to Evaluate the Response to Treatment in Solid Tumors", Journal of the National Cancer Institute, Vol. 92, No. 3, February 2, 2000) (updated in 2009 (Eisenhauer EA et al., "New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)", European Journal of Cancer, Volume 45, Issue 2, 228-247)). In this context, in another embodiment, a cold tumor is one that involves less than a partial response ("PR"; i.e., either progressing ("PD", progressive disease) or remaining stable ("SD", stable disease)) following treatment with one or more immune checkpoint inhibitors administered according to the instructions in the immune checkpoint inhibitor's package insert. In another embodiment, a cold tumor is one that involves less than a partial response ("PR"; i.e., either progressing ("PD", progressive disease) or remaining stable ("SD", stable disease)) following treatment with one or more immune checkpoint inhibitors administered for at least 6 weeks, preferably at least 8 weeks, and more preferably at least 12 weeks. In another embodiment, the cold tumor is a breast cancer tumor that is progressive following anti-PD-L1 treatment.

[0060] In another embodiment, a cold tumor is a tumor associated with progressive disease ("PD") at least 6 weeks, preferably at least 8 weeks, and more preferably at least 12 weeks after one or more immune checkpoint inhibitors administered according to the immune checkpoint inhibitor package insert.

[0061] In another embodiment, a cold tumor is a tumor that becomes unresponsive to one or more immune checkpoint inhibitors, i.e., a tumor that previously responded to treatment with one or more immune checkpoint inhibitors but has become unresponsive.

[0062] In another embodiment, a cold tumor may also be defined as a tumor that recurs (i.e., has a diminished response to treatment) after an initial response to one or more immune checkpoint inhibitors, where the latter is dosed according to the instructions in the immune checkpoint inhibitor's package insert.

[0063] In another embodiment, the cold tumor is a tumor classified as a cold tumor by a clinician. A specific, non-limiting example of a cold tumor is colorectal cancer tumor with low microsatellite instability (MSI-low; Sinicrope, F. “The Role of Microsatellite Instability Testing in Management of Colorectal Cancer”, Clinical Advances in Hematology & Oncology Volume 14, Issue 7 July 2016). Such tumors are known to be non-responsive to immune checkpoint inhibitors and can be identified as cold tumors without first treating them with one or more immune checkpoint inhibitors to establish non-responsiveness.

[0064] As mentioned above, "cold tumors" respond poorly to treatment with immune checkpoint inhibitors, such as anti-PD-1 or anti-CTLA-4 antibodies. The rationale behind this phenomenon may be that if the immune system is not involved in the respective tumor, blocking immune checkpoints with a given inhibitor cannot change anything for the better.

[0065] In particular, a subset of melanoma, colorectal cancer or tumors, pancreatic cancer or tumors, glioblastoma, ovarian cancer or tumors, and prostate cancer or tumors are known to be cold tumors.

[0066] The present inventors (i) a binding protein-toxin fusion comprising one or more anthracycline toxin moieties fused to a binding protein, and (ii) showing that such cold tumors can be effectively treated using treatments involving immune checkpoint inhibitors;

[0067] This finding was surprising and unexpected from findings made with immune checkpoint inhibitors, such as anti-PD-1, anti-CTLA-4 or anti-PD-L1 therapies, and with anthracycline-containing binding protein-toxin fusions. Without wishing to be bound by theory, anthracycline-containing binding protein-toxin fusions can sensitize cold tumors to immune checkpoint therapy, i.e., transform them into hot tumors, so that they can be effectively treated with immune checkpoint inhibitors.

[0068] Preferably, the two different agents are administered to the patient no more than a maximum of four weeks apart.

[0069] In one embodiment, a binding protein-toxin fusion comprising one or more anthracycline toxins is administered prior to the immune checkpoint inhibitor.

[0070] According to one embodiment, the at least one anthracycline toxin moiety is PNU-159682, which is disclosed in (Quintierei et al., 2005).

[0071] According to another embodiment, at least one anthracycline toxin moiety is a derivative of the anthracycline PNU-159682 having the formula (i): [ka] The toxin is fused to the binding protein at its undulating portion via a linker.

[0072] PNU-derived anthracyclines and their use in antibody-drug fusions are potentially disclosed in commonly assigned WO2016102679, the contents of which are incorporated herein by reference.

[0073] According to another group of embodiments, the immune checkpoint inhibitor is at least one selected from the group consisting of: ·Anti-PD-1 ·Anti-PD-L1 ·Anti-PD-L2 ·Anti-CTLA-4 ·Anti-LAG3 Anti-CD40 or anti-CD40L ·Anti-TIM3, Anti-OX40 or anti-OX40L (CD134 / CD134L), ·Anti-CD112 ·Anti-CD155 ·Anti-B7-H3 Anti-B7-H4 ·Anti-IDO1 ·Anti-IDO2 ·Anti-TDO2 ·Anti-TIGIT Anti-GITR, and / or Anti-galectin-9.

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[0084] Among the above checkpoint inhibitors, the following are compounds or monoclonal antibodies that bind or antagonize: PD-1 PD-L1 CTLA-4, and / or B7 are particularly preferred, with PD-1 and PD-L1 being the most preferred targets.

[0085] According to another group of embodiments, the fusion comprises, at the wavy line, the linker structure X-L1-L2-L3-Y, where L1-L3 represent a linker and two of L1-L3 are mandatory, and where X and Y further each represent one or more optional linkers.

[0086] According to another group of embodiments, the linker structure comprises as L2 an oligoglycine peptide (Gly) linked directly or by means of another linker L1 to the anthracycline derivative. n where n is an integer between ≧1 and ≦21, preferably 2 to 5. In one specific embodiment (where Streptococcus pyogenes sortase A is used, see below), oligoglycine (Gly) n Optionally, oligoalanine (Ala) n It is important to understand that the nucleotide sequence may be replaced by a mixed Gly / Ala peptide or a mixed Gly / Ala peptide.

[0087] According to another group of embodiments, oligoglycine peptides (Gly) n is attached to the anthracycline derivative of formula (i) by means of an alkylenediamino linker (EDA) designated L1, which is attached to the anthracycline derivative by means of a first amide bond, which in turn is attached to the carboxy terminus of an oligoglycine peptide by means of a second amide bond, said alkylenediamino linker and oligoglycine peptide fusion having the following formula (ii): [ka]

[0088] wherein the wavy line indicates the bond to the anthracycline derivative of formula (i), In the formula, m is an integer of 1 or more and 11 or less, preferably 2 to 5, and n is an integer of 1 or more and 21 or less, and preferably an integer of 2 to 5. In one specific embodiment (where Streptococcus pyogenes sortase A is used, see below), oligoglycine (Gly) n is optionally oligoalanine (Ala) n It is important to understand that it can be replaced by

[0089] According to another group of embodiments, oligoglycine peptides (Gly) n is attached directly or by means of another linker L1 to ring A of the anthracycline derivative of formula (ii) (wherein ring A of the anthracycline core is as shown in Shi et al., 2009). In one specific embodiment (where Streptococcus pyogenes sortase A is used, see below), oligoglycine (Gly) n is optionally oligoalanine (Ala) n It is important to understand that it can be replaced by

[0090] According to another group of embodiments, oligoglycine peptides (Gly n ) is fused to the anthracycline derivative of formula (i) by means of an alkylene amino linker (EA) designated L1, which is fused to the carboxy terminus of an oligoglycine peptide by means of an amide bond, said fusion of alkylene amino linker with the oligoglycine peptide having the following formula (iii): [ka]

[0091] wherein the wavy line indicates the bond to the anthracycline derivative of formula (i), In the formula, m is an integer of 1 or more and 11 or less, preferably 2 to 5, and n is an integer of 1 or more and 21 or less, and preferably an integer of 2 to 5. In one specific embodiment (where Streptococcus pyogenes sortase A is used, see below), oligoglycine (Gly) n is optionally oligoalanine (Ala) n It is important to understand that it can be replaced by

[0092] According to another group of embodiments, the linker structure L3 comprises a peptide motif resulting from specific cleavage of a sortase enzyme recognition motif.

[0093] The sortase enzyme recognition motif serves as a tag for the so-called sortase enzyme mediated antibody fusion (SMAC technology), which is enablingly disclosed in WO2014140317, assigned to the same applicant. The contents of this publication are incorporated herein by reference. In this technique, one molecule has such a recognition motif, and the other molecule has an oligoglycine peptide (Gly). n The enzyme sortase fuses two molecules having the following structure: In one specific embodiment (when S. pyogenes sortase A is used, see below), oligoglycine (Gly) n is optionally an oligoalanine (Ala) n It is important to understand that it can be replaced by

[0094] According to another group of embodiments, the sortase enzyme recognition motif comprises at least one of the following amino acid sequences: LPXTG, LPXAG, LPXSG, LAXTG, LPXTA, or NPQTN, where X is any possible amino acid sequence.

[0095] According to another group of embodiments, the resulting linker has at least one of the following sequences: -LPXTG n -, -LPXAG n -, -LPXSG n -, -LAXTG n -, -LPXTG n -, -LPXTA n -,or -NPQTG n -, Here, G n is an oligo- or polyglycine, where n is an integer from ≧1 to ≦21; A n is an oligo- or polyalanine, where n is an integer from ≧1 to ≦21; and X is any possible amino acid sequence.

[0096] According to another group of embodiments, the anthracycline derivative is attached to the carboxy terminus of the binding protein, or of at least one domain or subunit thereof, by means of one or more linkers.

[0097] According to another group of embodiments, the binding protein is linked by means of an amide bond to an oligoglycine peptide (Gly). n is attached to the amino terminus of In one specific embodiment (when S. pyogenes sortase A is used, see below), oligoglycine (Gly) n is optionally an oligoalanine (Ala) n It is important to understand that it can be replaced by

[0098] The sortase used is preferably sortase A, and in one embodiment is sortase A from Streptococcus pyogenes or Staphylococcus aureus. In one embodiment, an engineered sortase A is used that still recognizes the sortase A recognition motif. In one embodiment, the engineered sortase A is derived from Streptococcus pyogenes sortase A, and in one other embodiment, the engineered sortase A is derived from Staphylococcus aureus sortase A.

[0099] According to another group of embodiments, the binding protein is at least one selected from the group consisting of an antibody, an antibody-based binding protein, a modified antibody format that retains target binding ability, an antibody derivative or fragment that retains target binding ability, an alternative scaffold and / or an antibody mimetic.

[0100] Of the above, antibodies are the most preferred type of binding protein.

[0101] The terms "antibody," "antibody-based binding protein," "modified antibody formats which retain target binding ability," and "antibody derivatives or fragments which retain target binding ability" refer to polypeptide chains that exhibit strong monovalent, divalent or multivalent binding to a given antigen, epitope or epitopes. The antibodies, antibody-based binding proteins and antigen-binding fragments used in the present invention can be generated using any suitable technique, such as hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries, or a combination thereof. The antibodies, antibody-based binding proteins, and antigen-binding fragments of the present invention include intact antibodies and antibody fragments, or antigen-binding portions of intact antibodies and which retain the ability to bind to cognate antigens. Unless otherwise stated herein, all peptide sequences, including all antibody and antigen-binding fragment sequences, are referred to in N→C order.

[0102] An intact antibody typically comprises at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) inter-connected by disulfide bonds. The recognized immunoglobulin genes which encode antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Each heavy chain of an antibody comprises a heavy chain variable region (V H ) and the heavy chain constant region. In IgG, the heavy chain constant region is C H1 , C H2 and C. H3 Each light chain is composed of three regions: L ) and the light chain constant region. The light chain constant region consists of one domain, C L It is composed of: The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the first component (Clq) of the classical complement system. Monoclonal antibodies (mAbs) consist of identical antibody molecules.

[0103] V of antibody H and V L The regions can be further subdivided into regions of hypervariability interspersed with more conserved framework regions (FR), also called complementarity determining regions (CDRs). Each V H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The locations of the CDR and FR regions and the numbering system as used herein are defined, for example, as the IMGT system (Feffane MP et al., 2015).

[0104] The antibodies, antibody-based binding proteins, and antigen-binding fragments of the present invention also include single chain antibodies. The term "single chain antibody" generally refers to a V in polypeptide bond linked via a spacer peptide. H Domains and V L It refers to a polypeptide that contains a domain and may contain additional domains or amino acid sequences at the amino and / or carboxyl termini. For example, a single chain antibody can include a tether segment for linking to the encoding polynucleotide. As an example, a single chain variable fragment (scFv) is a single chain antibody. V of Fv fragments encoded by separate genes L and V H In contrast to domains, scFvs have two domains linked (eg, by recombinant methods) by a synthetic linker. This means that V L Area and V H This allows the two domains to be produced as a single protein chain that pairs to form monovalent molecules.

[0105] Examples of antibody-based binding proteins are polypeptides in which the binding domains of the antibodies bind to other polypeptides or polypeptide domains (e.g., alternative molecular scaffolds), Fc regions, other functional or binding domains of other polypeptides, or antibodies resulting in molecules with additional binding properties (e.g., bispecific or multispecific proteins or antibodies). Such polypeptides may create arrangements of binding or functional domains not normally found in naturally occurring antibodies or antibody fragments.

[0106] Examples of antigen-binding fragments include: (i) Fab fragment, V L , V H , C L and C. H1 A univalent fragment consisting of a domain; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region; (iii) V H and C. H1 Fd fragment consisting of domains; (iv) V of a single arm of an intact antibody L and V H Fv fragment consisting of domains; (v) disulfide-stabilized Fvs (dsFvs), which have engineered interchain disulfide bonds between structurally conserved framework regions; (vi) V H or V L single domain antibodies consisting of domains (see, e.g., Ward et al., Nature 341:544-546, 1989); and (vii) independent complementarity determining regions (CDRs) as linear or cyclic peptides; Examples include:

[0107] Antigen-binding fragments of the invention also include single domain antigen-binding units having a camelid scaffold. Camelids include camels, llamas, and alpacas. Camelids produce functional antibodies that lack light chains. Heavy chain variable (V H ) domains fold autonomously and function independently as antigen-binding units. Its binding surface comprises only three CDRs compared to six CDRs in classical antigen-binding molecules (Fabs) or single-chain variable fragments (scFvs). Camelid antibodies are able to achieve binding affinities comparable to those of conventional antibodies.

[0108] The term "human antibody" refers to an antibody, antibody-based binding protein, or antigen-binding fragment that contains sequences derived from a human immunoglobulin, such that substantially all of the CDR regions are of human origin and substantially all of the FR regions correspond to those of human immunoglobulin sequences.

[0109] The terms "alternative scaffolds" and "antibody mimetics" refer to proteins that do not belong to the immunoglobulin family, and even non-proteins such as aptamers, or synthetic polymers. Some have an antibody-like β-sheet structure. Potential advantages of "antibody mimetics" and "alternative scaffolds" over antibodies include better solubility, greater tissue penetration, greater thermal and enzymatic stability, and relatively low production costs.

[0110] Several antibody mimetics can be provided in large libraries providing specific binding candidates for every possible target. As with antibodies, target-specific antibody mimetics can be developed by the use of high-throughput screening (HTS) techniques, as well as using established display technologies such as phage display, bacterial display, yeast or mammalian display. Antibody mimetics currently being developed include, for example: ankyrin repeat proteins (called DARPins), C-type lectins, A-domain proteins of Staphylococcus aureus, Transferrin, Lipocalin, Fibronectin type 10 III domain, Kunitz domain protease inhibitors, ubiquitin-derived binders (called affilins), Gamma crystallin-derived binders, cysteine ​​knots or knottins, Thioredoxin A scaffold-based binders, Nucleic acid aptamers, Artificial antibodies produced by molecular imprinting of polymers; Peptide libraries from bacterial genomes, SH-3 domain, Strayed body, The "A domain" of membrane receptors stabilized by disulfide bonds and Ca2+, CTLA4-based compounds, Fyn SH3, and Aptamers (oligonucleotide or peptide molecules that bind to specific target molecules) Includes:

[0111] The antibodies, antibody-based binding proteins and antigen-binding fragments of the invention may bind to any suitable target on a cancer cell or within the cancer cell environment of a tumor. In one embodiment, the antibodies, antibody-based binding proteins and antigen-binding fragments of the invention bind to targets on the surface of cancer cells. In preferred embodiments, the target is exclusively expressed on cancer cells or is overexpressed on cancer cells compared to healthy tissue. Applicants suggest that any target on a cancer cell to which the antibodies, antibody-based binding proteins and antigen-binding fragments of the invention bind and which the antibodies, antibody-based binding proteins and antigen-binding fragments of the invention, along with the anthracycline toxin moiety, are internalized into the cell, resulting in immunogenic cell death of the cell and / or infiltration of immune cells (e.g., T cells) into the tumor is a suitable target. One of skill in the art is able to identify such targets through review of the literature and experimentation.

[0112] In one embodiment, suitable targets include ROR1, CS-1, HER2, mesothelin and ROR2. In one embodiment, the antibodies, antibody-based binding proteins and antigen-binding fragments of the invention bind to ROR1.

[0113] According to a further embodiment of the invention, the binding protein included in the binding protein-toxin fusion is an antibody a) An antibody comprising a set of heavy / light chain complementarity determining regions (CDRs) contained in the heavy / light chain variable domain sequence pair set forth in the following pair of SEQ ID NOs: 1 and 2; 3 and 4, 5 and 6 and / or 20 and 21; b) An antibody comprising a set of heavy / light chain complementarity determining regions (CDRs) comprising the following SEQ ID NOs (in the order HCDR1; HCDR2; HCDR3; LCDR1; LCDR2 and LCDR3): 22, 23, 24, 25, 26, and 27, 28, 29, 30, 31, 32, and 33, 34, 35, 36, 37, 38, and 39, and / or 40, 41, 42, 43, 44, and 45; c) an antibody comprising the heavy / light chain complementarity determining regions (CDRs) of b), provided that at least one of the CDRs has up to three amino acid substitutions relative to the respective SEQ ID NO:; and / or

[0114] d) an antibody comprising the heavy / light chain complementarity determining regions (CDRs) of b) or c), provided that at least one of the CDRs has ≧66% sequence identity to the respective SEQ ID NO:;

[0115] Here, the CDRs are embedded in a suitable protein framework such that they can bind to ROR1 with sufficient binding affinity.

[0116] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions have been described by Kabat et al. (1977), Kabat et al. (1991), Chothia et al. (1987) and MacCallum et al. (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited descriptions are set forth in the Table below as a comparison. Please note that because CDR definitions vary in different cases, this numbering may differ from the CDRs actually disclosed in the enclosed sequence listing.

[0117] [Table 2]

[0118] As used herein, the term "framework", when used in reference to an antibody variable region, is taken to mean all amino acid residues outside the CDR regions in the variable region of an antibody. Thus, a variable region framework is between about 100-120 amino acids in length, but is intended to refer only to amino acids outside the CDRs.

[0119] As used herein, the term "capable of binding to target X with sufficient binding affinity" means that each binding domain has a binding affinity to the target, K D 10 -4 It should be understood to mean the following: K D is the K between a protein binder and its antigen. off / K on is the equilibrium dissociation constant, which is the ratio of K D and affinity are inversely related. K D The value is related to the concentration of the protein binder (the amount of protein binder required for a particular experiment) and therefore the K D The lower the value (lower the concentration) and therefore the higher the affinity of the binding domain. The table below shows the K values ​​for typical monoclonal antibodies. DThe range of

[0120] [Table 3]

[0121] Preferably, the protein binder has up to two amino acid substitutions, more preferably up to one amino acid substitution.

[0122] Preferably, at least one of the CDRs has a sequence similar to that of the amino acid sequence of ≧67%; ≧68%; ≧69%; ≧70%; ≥71%;≥72%;≥73%;≥74%;≥75%;≥76%;≥77%;≥78%; ≧79%;≧80%;≧81%;≧82%;≧83%;≧84%;≧85%≧86%;≧87%; ≧88%;≧89%;≧90%;≧91%;≧92%;≧93%;≧94%;≧95%;≧96%; ≧97%; ≧98%; ≧99% sequence identity, and most preferably 100% to each of the SEQ ID NOs.

[0123] As used herein, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide) that does not contain the additions or deletions due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where an identical nucleic acid base or amino acid residue occurs in both sequences, yielding the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.

[0124] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percentage of "identity" refers to two or more sequences or subsequences that are the same sequence.

[0125] Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are identical (i.e., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a specified region, or, if not specified, over the entire sequence of the reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated regions as measured using one of the sequence comparison algorithms below, or by manual alignment and visual inspection.

[0126] The present disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein. Optionally, the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100-500 or 1000 or more nucleotides in length, or over the entire length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or the entire length of the reference sequence. For shorter amino acid sequences, for example, amino acid sequences of 20 amino acids or less, substantial identity exists if one or two amino acid residues are conservatively substituted in accordance with conservative substitutions as defined herein.

[0127] Preferably, at least one of the CDRs has a CDR sequence modification comprising: ·Affinity maturation ·Reduced immunogenicity

[0128] Affinity maturation is the process by which the affinity of an antibody is improved in vitro. Like its natural counterpart, in vitro affinity maturation is based on the principles of mutation and selection. It has been successfully used to optimize antibodies, antibody fragments, or other peptide molecules such as antibody mimetics. Random mutations within the CDRs are introduced using radiation, chemical mutagens, or error-prone PCR. Furthermore, genetic diversity can be increased by the chain shuffling method. Two or three rounds of mutation and selection using display methods such as phage display usually yield antibody fragments with affinities in the low nanomolar range. For principles, see Eylenstein et al. (2016), the contents of which are incorporated herein by reference.

[0129] The engineered antibody contains the CDR regions derived from the murine sequence grafted onto the V regions derived from the sequence, along with any necessary framework backmutations. Thus, the CDRs themselves may cause an immunogenic response when the humanized antibody is administered to a patient. Methods for reducing immunogenicity caused by CDRs are disclosed in Harding et al. (2010), the contents of which are incorporated herein by reference.

[0130] According to a further embodiment of the invention, the binding protein included in the binding protein-toxin fusion is an antibody a) A heavy chain / light chain variable domain (HCVD / LCVD) pair as set forth in the following SEQ ID NO pair: 1 and 2; 3 and 4, 5 and 6, and / or 20 and 21; b) the heavy chain / light chain variable domain (HCVD / LCVD) pair of a), provided that: the HCVD has ≥80% sequence identity to each SEQ ID NO; and / or · The LCVD has ≥80% sequence identity to each SEQ ID NO: c) a heavy / light chain variable domain (VD) pair of a) or b), wherein at least one of the HCV D or LCVD has up to 10 amino acid substitutions relative to its respective SEQ ID NO:; The protein binder is still capable of binding to ROR1 with sufficient binding affinity.

[0131] "Variable domain", when used in reference to an antibody or a heavy or light chain thereof, is intended to mean the portion of the antibody that confers antigen binding on the molecule and is not the constant region. The term is intended to include functional fragments thereof which maintain some or all of the binding functions of the entire variable region. Variable region-binding fragments include functional fragments such as Fab, F(ab)2, Fv, and single-chain Fv (scfv). Such functional fragments are well known to those of skill in the art, and thus, use of these terms in describing functional fragments of heterologous variable regions is intended to correspond to definitions well known to those of skill in the art. Such terms are described, for example, in Huston et al. (1993) or Pluckthun and Skerra (1990).

[0132] Preferably, HCVD and / or LCVD is ≧81% for each SEQ ID NO: ≧82%;≧83%;≧84%;≧85%;≧86%;≧87%;≧88%;≧89%; ≧90%;≧91%;≧92%;≧93%;≧94%;≧95%;≧96%;≧97%; ≧98%; ≧99%; or most preferably 100% sequence identity.

[0133] According to another embodiment of the invention, the binding protein has at least one amino acid substitution which is a conservative amino acid substitution.

[0134] As used herein, "conservative amino acid substitutions" have a smaller effect on antibody function than non-conservative substitutions. Although there are many ways to classify amino acids, they are often divided into six main groups based on their structure and the general chemical characteristics of their R groups.

[0135] In some embodiments, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include the following amino acids: Amino acids with basic side chains (e.g., lysine, arginine, histidine), Amino acids with acidic side chains (e.g. aspartic acid, glutamic acid), Amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), Amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), Amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and · Amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0136] Other conservative amino acid substitutions can occur across amino acid side chain families, such as the replacement of asparagine with aspartic acid to modify the charge of the peptide. Conservative changes can further include substitutions of chemically homologous non-natural amino acids (ie, synthetic non-natural hydrophobic amino acids for leucine, synthetic non-natural aromatic amino acids for tryptophan). According to a further embodiment of the invention, the binding protein has a target binding affinity for ROR1 that is at least 50% compared to that of an antibody according to the above description.

[0137] As used herein, the term "binding affinity" is intended to mean the strength of a binding interaction and thus includes both actual binding affinity as well as apparent binding affinity, which is the ratio of the association rate to the dissociation rate. Thus, conferring or optimizing binding affinity encompasses modifying either or both of these components to achieve the desired level of binding affinity. The apparent affinity can include, for example, the avidity of the interaction. For example, a bivalent heterovariable region binding fragment may exhibit altered or optimized binding affinity by virtue of its valency.

[0138] A suitable method for measuring the affinity of a binding agent is by surface plasmon resonance (SPR). The method is based on a phenomenon that occurs when surface plasmon waves are excited at a metal / liquid interface: light is directed to the side of the surface that is not in contact with the sample, where it is reflected, and SPR causes a decrease in the reflected light intensity at a specific combination of angle and wavelength. A biomolecular binding event causes a change in the refractive index at the surface layer, which is detected as a change in the SPR signal. The binding event can be either a binding association or dissociation between a receptor-ligand pair. The change in refractive index can be measured essentially instantaneously, thus allowing the determination of the individual components of the affinity constant. More specifically, the method comprises determining the association rate (k on ) and dissociation rate (k off ) to enable accurate measurement.

[0139] k on and k off Measuring the levels can be advantageous because it allows identification of altered or optimized variable regions that are more effective therapeutically. For example, an altered variable region or heteromeric binding fragment thereof may have a higher kA binding affinity, e.g., compared to a variable region and a heteromeric binding fragment that exhibits a similar binding affinity. on It may be more useful because it has value. Higher k on Molecules having this value are able to specifically bind to and inhibit their targets at a faster rate, thus conferring increased potency. Similarly, the molecules of the invention have lower k off It may be more effective because it shows value. A lower k because, once bound, the molecules dissociate more slowly from their targets. off Increased potency can be observed with molecules that have a rate. Although described with reference to the altered and optimized variable regions of the present invention (including heterologous variable region binding fragments thereof), the above methods for measuring association and dissociation rates are applicable to essentially any protein binder or fragment thereof to identify more effective binders for therapeutic or diagnostic purposes.

[0140] Another suitable method for measuring the affinity of a binding agent is through a surface and by FACS / Scatchard analysis.

[0141] Methods for measuring affinity, including association and dissociation rates, using surface plasmon resonance are well known in the art and can be found, for example, in Jonsson and Malmquist (1992) and Wu et al. (1998). Additionally, one instrument well known in the art for measuring binding interactions is the BIAcore 2000 instrument available from Pharmacia Biosensor (Uppsala, Sweden).

[0142] Preferably, the target binding affinity is ≧51%, ≧52%, ≧53%, ≧54%, ≧55%, ≧56%, ≧57%, ≧58%, ≧59%, ≧60%, ≧61%, ≧62%, ≧63%, ≧64%, ≧65%, ≧66%, ≧67%, ≧68%, ≧69%, ≧70%, ≧71%, ≧72%, ≧73%, ≧74%, ≧75%, ≧76%, ≧77%, ≧78%, ≧79%, ≧80%, ≧81%, ≧82%, ≧83%, ≧84%, ≧85%, ≧86%, ≧87%, ≧88%, ≧89%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98% and most preferably ≧99% compared to that of a control binding agent.

[0143] According to another embodiment of the invention, the binding protein competes with an antibody according to the above description for binding to ROR1. According to another embodiment of the invention, the binding protein binds to essentially the same or the same region on ROR1 as the antibodies described above.

[0144] As used herein, the term "competes for binding" is used in reference to one of the antibodies defined by the above sequences, meaning that it is a variant of the latter in terms of its activity in binding to the same target as the actual protein binder, or to a target epitope or domain or subdomain. The efficiency of binding (eg, kinetics or thermodynamics) may be the same as, or greater or less than, the efficiency of the latter. For example, the equilibrium binding constants for binding to a substrate may be different for the two antibodies.

[0145] Such binding competition can be suitably measured using competitive binding assays. Such assays are disclosed in Finco et al., 2011, the contents of which are incorporated herein by reference, and their meaning for interpretation of the claims is disclosed in Deng et al., 2018, the contents of which are incorporated herein by reference.

[0146] To test this property, suitable epitope mapping techniques are available, including, inter alia, ·X-ray co-crystallography and cryogenic electron microscopy (cryo-EM) Array-based oligo-peptide scanning Site-directed mutagenesis mapping High-throughput shotgun mutagenesis epitope mapping Hydrogen-deuterium exchange, and / or ·Cross-linking-coupled mass spectrometry

[0147] These methods are disclosed and discussed, inter alia, in Banik et al. (2010) and DeLisser (1999), the contents of which are incorporated herein by reference.

[0148] Working Example

[0149] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered as illustrative or exemplary and not restrictive, and the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprise" does not exclude other elements or steps and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims shall not be construed as limiting the scope.

[0150] All amino acid sequences disclosed herein are presented from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are presented 5'→3'.

[0151] Example 1 Generation of purified recombinant anti-human ROR1 and isotype control antibodies

[0152] Expression vector Antibody variable region coding regions were generated by total gene synthesis (GenScript) using MNFGLRLIFLVLTLKGVQC as leader sequence and appropriately combined with human IgH-γ1 and IgL-κ or IgL-λ constant regions in the expression vector pCB14, as applicable. This vector is a derivative of the episomal mammalian expression vector pCEP4 (Invitrogen) and contains an EBV origin of replication, encodes the EBV nuclear antigen (EBNA-1), allows extrachromosomal replication, and contains a puromycin selection marker in place of the original hygromycin B resistance gene.

[0153] Expression and purification : pCB14-based expression vectors were transfected into HEK293T cells using Lipofectamine® LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, Reinach, Switzerland, 15388100); after 1 day of incubation (37° C., 5% CO2, growth medium: Dulbecco's Modified Eagle's Medium (DMEM) High Glucose (4.5 g / L) with L-glutamine containing 10% (v / v) fetal calf serum (FCS), 100 IU / mL Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept, Allschwil, Switzerland)), cells were grown under selection conditions (2 μg / mL puromycin (Sigma-Aldrich, Buchs SG, Switzerland, P8833-25 mg / mL stock)). Cells were split and further grown (37°C, 5% CO2); upon reaching confluency, tissue culture dishes were coated with 20 μg / ml poly-L-lysine (Sigma-Aldrich, P1524) for 2 h at 37°C and then washed twice with PBS.

[0154] The cells were then trypsinized and split 1:3 onto poly-L-lysine coated plates. After reaching confluence again, cells were washed with PBS and then the medium was replaced with production medium (DMEM / F-12, Gibco / Thermo Fisher Scientific, 31330-03) supplemented with 1 μg / mL puromycin (Sigma, P8833), 100 IU / mL Pen-Strep-Fungizone (Bioconcept), 161 μg / mL N-acetyl-L-cysteine ​​(Sigma-Aldrich, A8199) and 10 μg / mL L-glutathione reduced (Sigma-Aldrich, G6529). Supernatants were harvested every 2 weeks, filtered (0.22 μm) to remove cells, and stored at 4°C until purification.

[0155] For purification, the filtered supernatant was loaded onto a PBS-equilibrated Protein A HiTrap column (GE Healthcare, Frankfurt am Main, Germany, 17-0405-01) or a JSR Amsphere™ Protein A column (JSR Life Sciences, Leuven, Belgium, JWT203CE) and washed with PBS; elution was performed with 0.1 M glycine, pH 2.5, on an AEKTA pure (GE Healthcare). Fractions were immediately neutralized with 1 M Tris-HCl buffer (pH 8.0) and analyzed for protein purity and integrity by SDS-PAGE. Protein-containing fractions were mixed and subjected to buffer exchange using an Amicon filtration unit (Millipore, Schaffhausen, Switzerland, UFC901008) to reach a dilution of 1:100 in PBS, and then sterile filtered using a low-retention filter (0.20 μm, Carl Roth, Karlsruhe, Germany, PA49.1).

[0156] The antibodies were transiently expressed in CHO cells by methods known in the art, the recombinant antibodies were purified by standard Protein A purification from CHO cell supernatants as known in the art, and the purity and integrity of the recombinant antibodies were analyzed by SDS-PAGE.

[0157] [Table 4]

[0158] Example 2 Formation of ADCs by fusing glycine-modified toxins with mAbs using SMAC Technology™

[0159] Sortase A Recombinant and affinity purified sortase A enzyme based on Staphylococcus aureus sortase A was produced in E. coli according to the techniques described in WO2014140317A1.

[0160] Generation of glycine-modified toxins SMAC technology (trademark) TM To generate the fusion ADC, a pentaglycine-EDA-PNU derivative (G5-EDA-PNU) was produced by Concortis (shown in Figure 1). The identity and purity of the pentaglycine-modified toxin were confirmed by mass spectrometry and HPLC, respectively. G5-EDA-PNU exhibited a purity of >95% as determined by HPLC chromatography.

[0161] Sortase-mediated antibody fusions The toxins were fused to the antibodies in Table 2 by incubating LPETG-labeled mAbs [10 μM] with glycine-modified toxins [200 μM] and 3 μM sortase A in the listed fusion buffer at 25° C. for 3.5 hours. The reaction was terminated by passage over a rProtein A GraviTrap column (BioRad). Bound fusions were eluted with 5 column volumes of elution buffer (0.1 M glycine, pH 2.5, 50 nM NaCl) and 1 column volume fractions were collected in tubes containing 25% v / v 1 M HEPES (pH 8) to neutralize the acid. Protein-containing fractions were pooled and formulated in the formulation buffer of Table 2 using a ZebaSpin desalting column.

[0162] ADC analysis Drug-to-antibody ratios (DAR) were assessed by reversed-phase chromatography performed on a Polymer Labs PLRP 2.1 mm x 5 cm, 5 μm column using a 25 min linear gradient between 0.05-0.1% TFA / HO and 0.04-0.1% TFA / CHCN run at 1 mL / min / 80 °C. Samples were first reduced by incubation with DTT at pH 8.0 for 15 min at 37°C.

[0163] The DARs measured by reverse phase chromatography are summarized in Table 2 below.

[0164] [Table 5] From these analyses it can be concluded that the SMAC Technology™ fusion proceeded with high efficiency.

[0165] Example 3 Generation of EMT-6 cells stably expressing human ROR1 Murine EMT-6 breast cancer cells were cultured at 37°C and 5% CO2 in DMEM complete (Dulbecco's modified Eagle's medium (DMEM) high glucose (4.5 g / L)) supplemented with 10% (v / v) fetal calf serum (FCS), 100 IU / mL Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept, Allschwil, Switzerland). Cells were engineered to overexpress ROR1 by transposition as follows: The cells were centrifuged (6 min, 290 × g, 4 °C) and then resuspended in RPMI-1640 medium (5 × 10 6 The cells were resuspended in 100 mM NaCl (100 mM MgCl2) at 37 °C for 24 h. Next, 400 μL of the cell suspension was added to 400 μL of RPMI containing 13.3 μg of either the transposable vector pPB-PGK-Puro-ROR1, which leads to the co-expression of full-length ROR1 (NP_005003.2) and a puromycin resistance gene, and 6.6 μg of the transposase-containing vector pCDNA3.1_hy_mPB. The DNA / EMT-6 cell mixture was transferred to an electroporation cuvette (0.4 cm-gap, 165-2088, BioRad, Cressier, Switzerland) and electroporated using a Biorad Gene Pulser II with a capacitance extender at 300 V and 950 μF. The cells were then incubated at room temperature for 5–10 min. After incubation, cells were centrifuged at 290 × g for 6 min in an Eppendorf 5810R centrifuge, washed once, and subsequently resuspended in complete DMEM before being incubated at 37 °C in a humidified incubator with a 5% CO atmosphere. One day after electroporation, cell pools stably expressing human ROR1 were selected by adding 3 μg / mL puromycin (Sigma-Aldrich, P8833).

[0166] Single-cell clones expressing ROR1 were derived from antibiotic-selected EMT-6-ROR1 cells. In short, After trypsinization, 10 6 The cells are centrifuged in FACS tubes and The resulting pellet was resuspended in buffer (PBS containing 2% (v / v) FCS). The cells were then incubated with anti-ROR1 antibody 2A2 for 30 min (4° C., final concentration 2 μg / mL), followed by centrifugation and washing. Cells were then resuspended as described above and incubated in the dark (30 min, 4° C.) with anti-human IgG antibody (Fcγ specific) PE (eBioscience, Vienna, Austria, 12-4998-82) diluted 1:250, washed once in buffer, and kept on ice until single-cell sorting of antigen-expressing cells by FACS using a FACSAriaII instrument (BD Biocsiences, San Jose, USA).

[0167] Example 4 In vivo evaluation of anti-ROR1 ADCs in the EMT-6-ROR1 syngeneic breast tumor model 1x10 6 EMT-6-ROR1 clone 14 tumor cells were dissolved in 100 μl of PBS and orthotopically implanted into the mammary fat pad of each BALB / c mouse.

[0168] The average tumor volume is approximately 30-80 mm 3 On day 0 (D0), when tumor size (measured by calipers) was reached, mice were block randomized into groups of 8 mice according to tumor size. Next, mice were treated once with different doses (0.25 mg / kg, 0.5 mg / kg, and 1 mg / kg) of ADC to determine the suboptimal dose at which the ADC produced only a partial antitumor response (Figure 3). Following this, the same model was set up with treatment with or without low dose ADCs, including isotype-matched control ADCs according to Table 3, and anti-CTLA4 immune checkpoint inhibitor mAb 9D9 at 10 mg / kg. Mice were monitored for at least 40 days and euthanized in the event of excessive tumor burden or signs of distress. The immune checkpoint inhibitor 9D9 is an inhibitor of murine CTLA-4, formulated in PBS and purchased from BioXcell, West Lebanon, New Hampshire, US.

[0169] [Table 6] Tumor volumes were determined based on twice-weekly caliper measurements of tumor size. Group median tumor volumes were calculated taking into account the values ​​of animals surviving on a given day. Additionally, tumor volumes of animals euthanized due to high tumor burden were carried forward using the last-observation-carried-forward (LOCF) method as long as the group mean / median tumor burden increased.

[0170] FIG. 4 shows the change in tumor volume for individual mice, as well as the median change in tumor volume. (A) Vehicle control; (B) Ac10-G5-PNU isotype control; (C)XBR1-402-G5-PNU, (D) Ac10-G5-PNU isotype control + anti-mouse CTLA-4 antibody 9D9; (E) XBR1-402-G5-PNU + anti-mouse CTLA-4 antibody 9D9; Changes in tumor volume for individual mice treated with

[0171] (F) Median group tumor volume change (by applying the Last-Observation-Carried-Forward (LOCF) method) for vehicle control, Ac10-G5-PNU isotype control, and XBR1-402-G5-PNU groups.

[0172] (G) Median group tumor volume change for vehicle control, Ac10-G5-PNU isotype control and anti-mouse CTLA-4 antibody 9D9, and XBR1-402-G5-PNU and anti-mouse CTLA-4 antibody 9D9 groups (with application of the Last-Observation-Carried-Forward (LOCF) method).

[0173] As shown by the results in the figure, the combination of XBR1-402-G5-PNU and the anti-mouse CTLA-4 antibody 9D9 provides an enhanced anti-tumor response compared to individual treatment with the ADC alone at 0.25 mg / kg. Furthermore, panel D shows that the anti-mouse CTLA-4 antibody 9D9 exhibits some anti-tumor efficacy compared to vehicle (panel A) and isotype control (panel B), supporting that the EMT-6-ROR1 tumor model can be characterized as a hot tumor model.

[0174] Furthermore, all surviving mice in group 11 (and not in the other groups? And maybe it's important to show?) received 1 × 10 6Upon rechallenge (day 102) with EMT-6-ROR1 clone 14 tumor cells implanted into the mammary fat pad without prior injection, mice were protected from tumor growth (not shown), suggesting that only the combination of ADC and checkpoint inhibitor generated a protective antitumor immune response.

[0175] Example 5 Generation of CT-26 and B16-F10 cells stably expressing human ROR1 Murine CT-26 colon carcinoma cells and B16-F10 ("B16") melanoma cells (both from ATCC) were cultured at 37°C and 5% CO2 in RPMI medium containing 10% (v / v) fetal calf serum (FCS), 100 IU / mL Pen-Strep-Fungizone and 2 mM L-glutamine (all from Bioconcept, Allschwil, Switzerland). Cells were engineered to overexpress ROR1 by transplantation as follows: The cells were centrifuged in an Eppendorf 5810R centrifuge (6 min, 290 × g, 4 °C) and resuspended in RPMI medium (5 × 10 6 The cells were then resuspended at 100 µL (cells / ml). Then, 400 µL of the cell suspension was added to 400 µL of RPMI containing 13.3 µg of the transferase vector pPB-PGK-Puro-ROR1 (leading to the co-expression of full-length ROR1 (NP_005003.2) and a puromycin resistance gene) and 6.6 µg of the transferase-containing vector pcDNA3.1_hy_mPB. The DNA / CT-26 or B16 cell mixture was transferred to an electroporation cuvette (0.4 cm-gap, 165-2088, BioRad, Cressier, Switzerland) and electroporated using a Biorad Gene Pulser II with a capacitance extender of 300 V and 950 μF. The cells were then incubated at room temperature for 5-10 minutes. After incubation, cells were centrifuged at 290×g for 6 min, washed once, and then resuspended in complete RPMI before incubation at 37° C. in a humidified incubator with a 5% CO atmosphere. One day after electroporation, cell pools stably expressing human ROR1 were selected by adding 3 μg / mL puromycin (Sigma-Aldrich, P8833).

[0176] Single-cell clones of CT-26 expressing ROR1 were derived from antibiotic-selected cells. In short, After trypsinization, 10 6 The cells are centrifuged in FACS tubes and The resulting pellet was resuspended in buffer (PBS containing 2% (v / v) FCS). The cells were then incubated with the anti-ROR1 antibody XBR1-402 for 30 min (4° C., final concentration 2 μg / mL); This was followed by centrifugation and washing. The cells were then resuspended as before and incubated with anti-human IgG antibody (Fcγ specific) PE (eBioscience, Vienna, Austria, 12-4998-82) at a 1:250 dilution in the dark (30 min, 4° C.), washed once in buffer, and kept on ice until single-cell sorting of antigen-expressing cells by FACS using a FACSAria II instrument (BD Biocsiences, San Jose, USA). FIG. 5(A) shows the FACS staining results of engineered CT-26 clone 3. Expression of ROR1 in the B16 pool used in the following experiments was also determined by FACS (FIG. 5(B)).

[0177] Example 6 hROR1-overexpressing CT26 colon cancer mouse model treated with anti-ROR1 ADC and immune checkpoint inhibitor combination CT26 establishes warm-to-hot tumors in mouse models, meaning that they respond to immune checkpoint inhibitors at high enough doses (Mosely, S et al., 2017). As shown in Table 4, each experimental group contained eight 6- to 8-week-old female BALB / c mice. 1x10 in 100 µL PBS 6 CT-26-ROR1 clone 3 tumor cells (from Example 5) were injected into the flank of each mouse. one side The median tumor volume was approximately 30 to 100 mm. 3 On day 0 (D0), when tumor size was reached (by caliper), mice were randomized into groups according to tumor size. Mice were then treated according to Table 4, 3 times per week for 42 days or until tumors reached 1500 mm 3 The temperature was monitored (by caliper) until it reached RPM1-14 (BioXcell, West Lebanon, New Hampshire, US) is an inhibitor of mouse PD-1 and was formulated in PBS. This immune checkpoint inhibitor was previously administered below its effective dose as monotherapy, as determined by Oncotest (data not shown).

[0178] [Table 7] FIG. 6 shows the tumor volume change for individual mice in each group: (A) Group 1, vehicle control (untreated); (B) Group 2 isotype control group; (C) Group 3 anti-ROR1 ADC huXBR1-402-17-G3-PNU; (D) Group 4 isotype control + immune checkpoint inhibitor anti-mouse PD-1 antibody; (E) Group 7 anti-ROR1 ADC huXBR1-402-17-G3-PNU + immune checkpoint inhibitor anti-mouse PD-1 antibody. As shown in panel C, it is emphasized that the targeted ADC was administered at a dose significantly lower than that required for a complete response as monotherapy. The results suggest strong synergy between the evaluated immune checkpoint inhibitors and the targeted PNU derivative-containing ADCs.

[0179] Example 7 hROR1-overexpressing B16 melanoma mouse model treated with anti-ROR1 ADC and immune checkpoint inhibitor combination B16 establishes poorly immunogenic (cold) tumors in mouse models (Celik, C et al., 1983), meaning that they do not respond to immune checkpoint inhibitors when administered at doses within a reasonable range, i.e., up to approximately 50-100 μg / mouse (Grosso, J et al., 2013). Each experimental group according to Table 7 contained 10 female BL6 / 6NRj mice aged 8-9 weeks. 2x10 in 100 μL of Hanks' Balanced Salt solution (HBSS, Thermo Fischer) 5 B16-ROR1 tumor cells (from Example 5) were injected intravenously into each mouse on day 0 (D0). Mice were randomly assigned to groups by simple random allocation and treated according to Table 5 and monitored 5 days per week for 12-19 days.

[0180] [Table 8] On the final day of monitoring, mice were euthanized with CO2, and whole lungs were subsequently harvested and fixed in Bouin's solution (Labforce Bio-Optica). Experimentally induced metastases on each lung surface (Saxena M. et al., 2013), representing B16-ROR1 colonies, were visually counted and each was assessed under blinded conditions for assignment to one of three bins (<1 mm, 1-2 mm, >2 mm in diameter). The volume of the colonies on each lung was then determined according to the following formula (approximating each colony as a sphere):

[0181] Colony volume (mm 3 ) = counts (spot diameter < 1mm) x 0.004mm 3 +Count (spot diameter 1-2mm) x 0.21mm 3 + Count (diameter > 2mm spots) x 0.485mm 3

[0182] Statistical analysis was performed using GraphPad Prism and unpaired T-tests of means, with p-values ​​<0.05 considered statistically significant (*) and p-values ​​≥0.5 considered statistically not significant (ns).

[0183] FIG. 7 shows the colony volumes of the different treatment groups in Table 5. These results suggest strong synergy between the evaluated immune checkpoint inhibitors and the targeted PNU derivative-containing ADCs in poorly immunogenic ("cold") tumors without significant effects of immune checkpoint inhibitors.

[0184] Example 8 hROR1-overexpressing B16 melanoma mouse model treated with anti-ROR1 ADC The experimental groups according to Table 6 included 8-9 week old female BL6 / 6NRj. 10 in 100 μL of Hanks' Balanced Salt solution (HBSS, Thermo Fischer) 6 B16-ROR1 tumor cells (from Example 5) were implanted into one flank of each mouse. The median tumor volume was approximately 250 mm 3 On day 0 (D0), when the mean (by caliper) was reached, mice were randomized into groups according to tumor size in Table 6. Mice were then treated according to Table 6 and euthanized 24 hours after dosing, followed by tumor extraction.

[0185] [Table 9] Tumors were snap frozen in optimal cutting temperature compound (oct, Fischer Scientific). Cryosections were stained with primary commercial anti-mouse CD4 and anti-mouse CD8 antibodies (GK1.5 BioLegend 100402 and 53-6.7 BioLegend 100702, respectively). Detection was performed using an anti-rat antibody (AlexaFluor488, Invitrogen A21208).

[0186] FIG. 8 shows representative images of CD4 and CD8 staining of hROR1 (human ROR1)-overexpressing B16 tumor frozen sections from (A) untreated control mice, and (B) three mice treated with ADC (once at 4 mg / kg) according to Table 6. According to FIG. 8(A), B16 tumors naturally contain low levels of CD4+ and CD8+ cells, maintaining the status of B16 tumors as cold tumors. FIG. 8(B) shows that treatment with an ADC according to the invention increases the number of CD4+ and CD8+ cells within the tumor.

[0187] Furthermore, hROR1 (human ROR1)-overexpressing CT-26 and B-16 tumors were mechanically and enzymatically digested and subjected to FACS staining for viable cells and immune cell markers to confirm the T cell population. The FACS staining scheme reported in Table 7 was used in combination with the LIVE / DEAD™ fixed near-infrared dead cell staining kit for 633 or 635 nm excitation staining reagents (ThermoFischer).

[0188] [Table 10] FIG. 9 shows the activation status of CD4 (panel A) and CD8 (panel B) positive T cell populations in two tumor models (untreated), confirming the "cold" nature of the hROR1 (human ROR1)-overexpressing B-16 tumor model.

[0189] References ·Galon J et al., J Pathol 2014; 232: 199-209 ·Pages F et al., J Clin Oncol 2009; 27: 5944-5951. ·Angell HK, Galon J, Curr Opin Immunol 2013; 25: 261-267. ·Galon et al. Immunity 2013; 39: 11-26. ·Bu X et al., Trends in Molecular Medicine (2016), Volume 22 , Issue 6 , 448 - 451 ·Shi et al., Org. Biomol. Chem., 2009,7, 3709-3722 ·Mosely, S. et al. Cancer Immunology Research, January 2017 ·Saxena M and Christofori G., Molecular Oncology 7, 2013, 283-296 ·Celik, C. et al. Cancer Res 43, 3507-3510, 1983 ·Grosso, J. et al. Cancer Immunity, 22, 13(5), 2013 ·U. Jonsson and M. Malmquist, Adv. Biosens., 1992, 2, 291 ·Wu et al. Proc. Natl. Acad. Sci. USA, 95:6037-6042 (1998) ·Banik, SSR; Doranz, BJ (2010). "Mapping complex antibody epitopes". Genetic Engineering & Biotechnology News. 3 (2): 25-8 ·DeLisser, HM (1999). Epitope mapping. Methods Mol Biol. 96. pp. 11-20 ·Finco et al, Comparison of competitive ligand-binding assay and bioassay for mats for the measurement of neutralizing antibodies to protein therapeutic s. J Pharm Biomed Anal. 2011 Jan 25;54(2):351-8. ·Deng et al., Enhancing antibody patent protection using epitope mapping info rmation MAbs. 2018 Feb-Mar; 10(2): 204-209 ·Huston et al., Cell Biophysics, 22:189-224 (1993); ·Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (19 90) ·Harding, The immunogenicity of humanized and fully human antibodies. MAbs. 2 010 May-Jun; 2(3): 256-265. ·Eylenstein, et al, Molecular basis of in vitro affinity maturation and funct ional evolution of a neutralizing anti-human GM-CSF antibody, mAbs, 8:1, 1 76-186 (2016) ·Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991) ·Chothia et al., J. Mol. Biol. 196:901-917 (1987) ·MacCallum et al., J. Mol. Biol. 262:732-745 (1996)

[0190] array The following sequences form part of the disclosure of this application. A WIPO ST 25 compliant electronic sequence listing is also provided with this application. For the avoidance of doubt, in the event of any discrepancy between the sequences in the table below and those in the electronic sequence listing, the sequences in the table shall be deemed correct. [Table 11] JPEG0007607556000025.jpg221170JPEG0007607556000026.jpg61170

Claims

1. For use in the treatment of neoplastic diseases characterized as cold tumors. (i) a binding protein-toxin fusion comprising one or more anthracycline toxin moieties fused to a binding protein; and (ii) immune checkpoint inhibitors; A pharmaceutical comprising the combination of The binding protein is at least one selected from the group consisting of an antibody, a modified antibody format that retains target binding ability, and / or a fragment that retains target binding ability; and The one or more anthracycline toxin moieties are derivatives of the anthracycline PNU-159682 having formula (i): 【Chemistry 1】 wherein the toxin is fused to the binding protein at the wavy line via a linker, and the immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG3, anti-CD40 or anti-CD40L, anti-TIM3, anti-OX40 or anti-OX40L (CD134 / CD134L), anti-CD112, anti-CD155, anti-B7-H3, anti-B7-H4, anti-IDO1, anti-IDO2, anti-TDO2, anti-TIGIT, anti-GITR, and / or anti-galectin-9; wherein the binding protein-toxin fusion and the immune checkpoint inhibitor are administered simultaneously or sequentially, in any order, to a patient.

2. 1. The use of an immune checkpoint inhibitor (ICI) for use in combination with a binding protein-toxin fusion comprising one or more anthracycline toxin moieties fused to a binding protein for the manufacture of a medicament for the treatment of a neoplastic disease characterized as a cold tumor, comprising The binding protein is at least one selected from the group consisting of an antibody, a modified antibody format that retains target binding ability, and / or a fragment that retains target binding ability; and The one or more anthracycline toxin moieties are derivatives of the anthracycline PNU-159682 having formula (i): 【Chemistry 2】 wherein the toxin is fused to the binding protein at the wavy line via a linker, and The immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG3, anti-CD40 or anti-CD40L, anti-TIM3, anti-OX40 or anti-OX40L (CD134 / CD134L), anti-CD112, anti-CD155, anti-B7-H3, anti-B7-H4, anti-IDO1, anti-IDO2, anti-TDO2, anti-TIGIT, anti-GITR, and / or anti-galectin-9.

3. The pharmaceutical product of claim 1, wherein the neoplastic disease characterized as a cold tumor is a tumor that is refractory, resistant to immune checkpoint inhibitor treatment, or recurrent after immune checkpoint inhibitor treatment.

4. The use according to claim 2, wherein the neoplastic disease characterized as a cold tumor is a tumor that is refractory, resistant to immune checkpoint inhibitor treatment, or recurrent after immune checkpoint inhibitor treatment.

5. 4. The pharmaceutical composition according to any one of claims 1 and 3, wherein the neoplastic disease characterized as a cold tumor is selected from the group consisting of: ·melanoma, - Colon cancer or colon tumors, Pancreatic cancer or tumor Glioblastoma, - ovarian cancer or tumor, and / or - Prostate cancer or tumor.

6. The use according to any one of claims 2 and 4, wherein the neoplastic disease characterized as a cold tumor is selected from the group consisting of: ·melanoma, - Colon cancer or colon tumors, Pancreatic cancer or tumor Glioblastoma, - ovarian cancer or tumor, and / or - Prostate cancer or tumor.

7. 6. The pharmaceutical agent of any one of claims 1, 3 and 5, wherein the binding protein binds to at least one target selected from the group consisting of: ・ROR1 ・CS1 ・HER2 Mesothelin (MN) and / or ・ROR2.

8. The use according to any one of claims 2, 4 and 6, wherein the binding protein binds to at least one target selected from the group consisting of: ・ROR1 ・CS1 ・HER2 Mesothelin (MN) and / or ・ROR2.

9. 8. The pharmaceutical agent of any one of claims 1, 3, 5 and 7, wherein the neoplastic disease characterized as a cold tumor is characterized as having an immunoscore of <1.

10. 9. The use according to any one of claims 2, 4, 6 and 8, wherein the neoplastic disease characterized as a cold tumor is characterized as having an immunoscore of <1.

11. The binding protein is a) an antibody comprising a set of heavy / light chain complementarity determining regions (CDRs) contained in the heavy / light chain variable domain sequence pairs set forth in SEQ ID NOs: 1 and 2; 3 and 4, 5 and 6, and / or 20 and 21; b) an antibody comprising a set of heavy / light chain complementarity determining regions (CDRs) comprising the following SEQ ID NOs (in the order HCDR1; HCDR2; HCDR3; LCDR1; LCDR2 and LCDR3); 22, 23, 24, 25, 26, and 27, 28, 29, 30, 31, 32, and 33, 34, 35, 36, 37, 38, and 39, and / or 40, 41, 42, 43, 44, and 45; c) an antibody comprising the heavy / light chain complementarity determining regions (CDRs) of b), with the proviso that at least one of the CDRs has up to three amino acid substitutions relative to the respective SEQ ID NO:; and / or d) an antibody comprising the heavy / light chain complementarity determining regions (CDRs) of b) or c), provided that at least one of the CDRs has ≧66% sequence identity to the respective SEQ ID NO:; And, wherein the CDRs are embedded in a suitable protein framework such that they can bind to ROR1 with sufficient binding affinity, Preferably, the binding protein is a) an antibody comprising a heavy / light chain variable domain (HCVD / LCVD) pair as set forth in SEQ ID NOs: 1 and 2; 3 and 4, 5 and 6 and / or 20 and 21; b) an antibody comprising the heavy chain / light chain variable domain (HCVD / LCVD) pair of a), provided that: HCVD has ≧80% sequence identity to each SEQ ID NO: and / or - an antibody, wherein the LCVD has ≥ 80% sequence identity to the respective SEQ ID NO; and / or c) an antibody comprising a heavy / light chain variable domain (VD) pair of a) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to its respective SEQ ID NO; And, the binding protein is still capable of binding to ROR1 with sufficient binding affinity; 10. A pharmaceutical product according to any one of claims 1, 3, 5, 7 and 9.

12. The binding protein is a) an antibody comprising a set of heavy / light chain complementarity determining regions (CDRs) contained in the heavy / light chain variable domain sequence pairs set forth in SEQ ID NOs: 1 and 2; 3 and 4, 5 and 6, and / or 20 and 21; b) an antibody comprising a set of heavy / light chain complementarity determining regions (CDRs) comprising the following SEQ ID NOs (in the order HCDR1; HCDR2; HCDR3; LCDR1; LCDR2 and LCDR3); 22, 23, 24, 25, 26, and 27, 28, 29, 30, 31, 32, and 33, 34, 35, 36, 37, 38, and 39, and / or 40, 41, 42, 43, 44, and 45; c) an antibody comprising the heavy / light chain complementarity determining regions (CDRs) of b), with the proviso that at least one of the CDRs has up to three amino acid substitutions relative to the respective SEQ ID NO:; and / or d) an antibody comprising the heavy / light chain complementarity determining regions (CDRs) of b) or c), provided that at least one of the CDRs has ≧66% sequence identity to the respective SEQ ID NO:; And, wherein the CDRs are embedded in a suitable protein framework such that they can bind to ROR1 with sufficient binding affinity, Preferably, the binding protein is a) an antibody comprising a heavy / light chain variable domain (HCVD / LCVD) pair as set forth in SEQ ID NOs: 1 and 2; 3 and 4, 5 and 6 and / or 20 and 21; b) an antibody comprising the heavy chain / light chain variable domain (HCVD / LCVD) pair of a), provided that: HCVD has ≧80% sequence identity to each SEQ ID NO: and / or - an antibody, wherein the LCVD has ≥ 80% sequence identity to the respective SEQ ID NO; and / or c) an antibody comprising a heavy / light chain variable domain (VD) pair of a) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to its respective SEQ ID NO; And, the binding protein is still capable of binding to ROR1 with sufficient binding affinity; 11. Use according to any one of claims 2, 4, 6, 8 and 10.

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