A humanized antibody or single-chain FV or FAB fragment capable of binding to a human common vascular endothelial and lymphatic endothelial receptor epitope -1 (CLEVER-1) and a pharmaceutical composition comprising the same.

Abstract

This invention relates to an agent and a humanized antibody or single-chain fv or fab fragment capable of binding to human clever-1 that recognizes a clever-1 epitope, wherein the epitope is discontinuous and comprises the sequences: pftvlvpsvssfssr and qeitvtfnqftk. This invention also relates to an agent capable of binding to a human clever-1 epitope for use in removing tumor- or antigen-induced immunosuppression. Furthermore, the invention relates to a pharmaceutical composition comprising the agent capable of binding to human clever-1 and a suitable excipient.

Description

1 / 50 “HUMANIZED ANTIBODY OR SINGLE-CHAIN ​​FV OR FAB FRAGMENT CAPABLE OF BINDING TO AN EPITOPE OF A LYMPHATIC ENDOTHELIAL AND VASCULAR ENDOTHELIAL RECEPTOR CLEVER-1 HUMAN PHARMACEUTICAL COMPOSITION AND COMPOSITION COMPRISING THE SAME FIELD OF THE INVENTION

[001] This invention relates to specific agents for the CLEVER-1 protein by recognizing a specific CLEVER-1 epitope and uses thereof. BACKGROUND OF THE INVENTION

[002] Publications and other materials used herein to clarify the background of the invention, and in particular, cases to provide additional details with respect to practice, are incorporated by reference.

[003] CLEVER-1 is a protein revealed in the WO document. 03 / 057130, Common Lymphatic Endothelial and Vascular Endothelial Receptor-1. It is a binding protein that mediates lymphocyte adhesion to the endothelium in both systemic and lymphatic vasculature. By blocking the interaction of CLEVER-1 and its lymphocyte substrate, it is possible to simultaneously control lymphocyte recirculation and migration, and related conditions such as inflammation, at the site of lymphocyte influx into and efflux from tissues. Document WO 03 / 057130 also reveals that CLEVER-1 mediates the binding of other types of leukocytes, such as monocytes and granulocytes, to HEV-type vessels. Thus, by blocking the interaction between CLEVER-1 and malignant tumor cells, it became possible to control metastasis, preventing malignant cells that bind to CLEVER-1 from being absorbed by lymphatic vessels and, therefore, preventing the spread of malignancy to the lymph nodes.

[004] CLEVER-1, that is, stabilin-1, was revised by Petition 870250108080, dated 11 / 26 / 2025, p. 7 / 116 2 / 50 Kzhyshkowska J. (2010), The Scientific World JOURNAL 10, 20392053. The suppression of Th1 lymphocytes by CLEVER-1 was recently revealed by Palani et al. (2016), Journal of Immunology 196: 115-123.

[005] The document WO 2010 / 122217 reveals a subtype of macrophages in tumors, in the placenta and in the blood of pregnant women. The macrophage subtype is defined as a CLEVER-1 positive macrophage and proposed as type 3 macrophage. By modulation, i.e., counterbalancing or stimulating, respectively, the CLEVER-1 receptor in this cell, an individual's immune system can be affected. Neutralization or downregulation of the receptor reduces malignant tumor size and / or malignant tumor growth. Stimulating or upregulating the receptor is useful in generating fetomaternal tolerance and preventing pregnancy complications.

[006] Mechanisms of tumor-associated macrophages (TAMs) are also revealed in the publication by Noy R. and Pollard JW."Tumor-Associated Macrophages: From Mechanisms to Therapy," published in Immunity 41, July 17, 2014, pp. 49-61. M2 macrophages predominate in human cancers and stimulate tumor growth, but these tumor-promoting macrophages can be modulated into tumor-inhibiting macrophages, also called M1 macrophages or pro-inflammatory macrophages, with the aim of slowing or preventing cancer growth. However, attempts to treat cancers with currently available therapies targeting TAMs have been found to be accompanied by undesirable side effects; for example, macrophage therapeutic approaches may have systemic toxicities or, paradoxically, promote tumor growth because they target all macrophages.

[007] Particularly preferential CLEVER-1 antagonist monoclonal antibodies 3-266 (DSM ACC2519) and 3-372 (DSM Petition 870250108080, dated 11 / 26 / 2025, page 8 / 116 3 / 50 ACC2520), both filed under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on August 21, 2001, at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, are disclosed in document WO 03 / 057130. OBJECTIVE AND SUMMARY OF THE INVENTION

[008] One objective of the present invention is to provide an agent capable of binding to a specific epitope of human CLEVER-1. In particular, it has been found that an agent capable of binding to a specific epitope of human CLEVER-1 can be used to activate macrophages to change their phenotype from M2 macrophages to M1 macrophages.

[009] Furthermore, an objective of the invention is to provide a humanized antibody or humanized single-chain Fv or Fab fragment for binding to human CLEVER-1 with increased binding activity compared to monoclonal antibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 21, 2001).

[0010] Therefore, the present invention provides an agent capable of binding to a human CLEVER-1 epitope, wherein the epitope is discontinuous and comprises the sequences: PFTVLVPSVSSFSSR (SEQ ID NO: 1) and QEITVTFNQFTK (SEQ ID NO: 2).

[0011] In particular, the present invention provides an agent capable of binding to human CLEVER-1, recognizing a CLEVER-1 epitope, wherein the epitope is discontinuous and comprises the sequences: PFTVLVPSVSSFSSR (SEQ ID NO: 1) and QEITVTFNQFTK (SEQ ID NO: 2), Petition 870250108080, dated 11 / 26 / 2025, p. 9 / 116 4 / 50

[0012] and the agent comprises the sequences of the complementarity determination regions (CDR) that bind to the so-called selected epitope sequences from the group consisting of TSGMGIG (SEQ ID NO: 7), HIWWDDDKRYNPALKS (SEQ ID NO: 8), HYGYDPYYAMDY (SEQ ID NO: 9), TASSSVSSSYLH (SEQ ID NO: 10), RTSNLAS (SEQ ID NO: 11) and HQYHRSPPT (SEQ ID NO: 12).

[0013] According to the invention, an agent capable of binding to A human CLEVER-1 antibody recognizing a CLEVER-1 epitope defined in the present application may be selected from the group consisting of an antibody, Fv fragment or single-chain fragment(s), peptide(s) or any other macromolecule that has a suitable affinity to bind to said epitope.

[0014] In one aspect, the present invention provides an agent capable of binding to human CLEVER-1 in an individual for use in removing tumor- or antigen-induced immunosuppression by modulating M2 macrophages into M1 macrophages, wherein the agent binds to a human CLEVER-1 epitope, which epitope is discontinuous and comprises the sequences: PFTVLVPSVSSFSSR (SEQ ID NO: 1) and QEITVTFNQFTK (SEQ ID NO: 2).

[0015] An agent according to the invention capable of binding to Human CLEVER-1 in TAMs, preferably specific epitope sequences in CLEVER-1, is suitable for use in the treatment or prevention of cancer by reducing the size of malignant tumors; reducing malignant tumor growth in an individual; and / or inhibiting the transmigration of cancer cells and the formation of metastases, wherein immunosuppression around malignant growth is achieved. Petition 870250108080, dated 11 / 26 / 2025, page 10 / 116 5 / 50 removed through modulation of M2 macrophages into M1 macrophages.

[0016] An agent according to the invention capable of binding to Human CLEVER-1, preferably with specific epitope sequences in CLEVER-1, is also suitable for use in the treatment of chronic infections in an individual, where immunosuppression against infectious antigens is removed by modulating M2 macrophages into M1 macrophages.

[0017] An agent according to the invention capable of binding to Human CLEVER-1, preferably with specific epitope sequences in CLEVER-1, is also suitable for use as a vaccine adjuvant, where immune suppression against vaccine antigens is removed by modulating M2 macrophages into M1 macrophages.

[0018] In another aspect, the invention provides a humanized antibody or single-chain Fv or Fab fragment capable of binding to a human CLEVER-1 epitope, wherein the epitope is discontinuous and comprises the sequences: PFTVLVPSVSSFSSR (SEQ ID NO: 1) and QEITVTFNQFTK (SEQ ID NO: 2),

[0019] and said antibody or single-chain Fv or Fab fragment comprise a) constant regions of the heavy chain and kappa light chain of human IgG4 and b) one or more of the following sequences of complementarity determination regions (CDRs)

[0020] i) of the heavy chain CDR 1: TSGMGIG (SEQ ID NO: 7), and / or CDR 2: HIWWDDDKRYNPALKS (SEQ ID NO: 8), and / or CDR 3: HYGYDPYYAMDY (SEQ ID NO: 9) and

[0021] ii) of the light chain CDR 1: TASSSVSSSYLH (SEQ ID NO: 10), and / or Petition 870250108080, dated 11 / 26 / 2025, p. 11 / 116 6 / 50 CDR 2: RTSNLAS (SEQ ID NO: 11) and / or CDR 3: HQYHRSPPT (SEQ ID NO: 12).

[0022] Another objective of the present invention is also to provide a pharmaceutical composition comprising the agent capable of binding to human CLEVER-1 or to the humanized antibody or to the single-chain Fv or Fab fragment according to the invention and a suitable excipient.

[0023] The present invention also provides a pharmaceutical composition comprising the agent capable of binding to human CLEVER-1 or to humanized antibody or to single-chain Fv or Fab fragment as defined above and an excipient suitable for use in removing tumor- or antigen-induced immunosuppression.

[0024] A pharmaceutical composition according to the invention is suitable for use in the treatment or prevention of cancer by reducing the size of malignant tumors; reducing malignant tumor growth in an individual; and / or inhibiting the transmigration of cancer cells and the formation of metastases. A pharmaceutical composition according to the invention is also suitable for use in the treatment of chronic infections in an individual or for use as an adjuvant to a vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figures 1a and 1b illustrate the heat map representation of the results obtained for antibody 3-266 and antibody AK FUMM 9-11.

[0026] Figure 2 illustrates the heat map representation of the results obtained for the FU-HI-3-372 antibody.

[0027] Figure 3 schematically illustrates the organization of the CLEVER-1 position domain of the identified linkage motifs.

[0028] Figure 4 illustrates the separation in a 1% agarose gel of Petition 870250108080, dated 11 / 26 / 2025, page 12 / 116 7 / 50 hybridoma RT-PCR products 3-372.

[0029] Figure 5 illustrates the SDS-PAGE gel, stained with blue. Coomassie, from chimeric IgG4 3-372 purified in protein A.

[0030] Figure 6 illustrates the CLEVER-1 competition ELISA.

[0031] Figure 7 illustrates the antitope vector diagram of pANT.

[0032] Figure 8 illustrates SDS-PAGE gel stained with blue Coomassie purified antibodies with selected protein A.

[0033] Figure 9 illustrates the CLEVER-1 competition ELISA.

[0034] Figure 10A shows the results of the determination of HLA-DR expression from CD14-positive cells and Figure 10B shows results of soluble TNF-alpha measured from the culture medium using a TNF-alpha ELISA kit (Invitrogen).

[0035] Figure 11A shows TAM repolarization in syngeneic E1771 breast carcinomas after administration of an antibody that binds to CLEVER-1 and Figure 11B shows increased TNF-alpha secretion in TAMs from syngeneic E0771 breast carcinoma after administration of an antibody that binds to CLEVER-1.

[0036] Figure 12 illustrates that the binding of CLEVER-1 to antibodies 9-11 and 3-372 promotes opposite effects on mTOR and c-Jun signaling in human peripheral blood monocytes. DETAILED DESCRIPTION OF THE INVENTION Terms

[0037] The term “an agent capable of binding to an epitope of "Human CLEVER-1" refers to agents including antibodies and fragments thereof, peptides or similar, that are capable of binding to specific epitope sequences defined in this application. The agent may also be any other macromolecule possessing a suitable affinity for binding to said epitope.

[0038] The term “an antibody or a fragment thereof” is used in the broadest sense to cover an antibody or a fragment of Petition 870250108080, dated 11 / 26 / 2025, page 13 / 116 8 / 50 even though it is capable of binding the CLEVER-1 molecule in an individual. Specifically, it should be understood that it includes chimeric, humanized or primatized antibodies, as well as antibody fragments and single-chain antibodies (e.g., Fab, Fv), provided they exhibit the desired biological activities.

[0039] The term humanized antibody refers to any antibody in which the constant regions of non-human antibodies have been wholly replaced by the human form of the constant regions and at least parts of the variable regions of non-human antibodies, excluding the three loops of amino acid sequences outside each variable region that bind to the target structure, have been wholly or partially replaced with corresponding parts of human antibodies. Thus, in particular, any antibody named by the nomenclature scheme for the International Nonproprietary Names (INN) of the World Health Organization or for the United States Adopted Names (USAN) for pharmaceutical products with sub-branches -xizu- or -zu- is called a humanized antibody.

[0040] The term variable domain, also called the Fv region, is the most important region for antigen binding. To be specific, the variable loops of the β chains, three in each light (Vl) and heavy (VH) chain, are responsible for antigen binding. These loops are referred to as complementarity-determining regions (CDRs).

[0041] The term single-chain Fv fragment or scFv refers to fragments that are obtained by linking the VH and VL domains via a ligand into a single polypeptide. The term humanized single-chain Fv fragment or scFv refers, in analogy with the definition of the term humanized antibody above, to any single-chain Fv fragment or scFv in which the constant regions originate Petition 870250108080, dated 11 / 26 / 2025, page 14 / 116 9 / 50 of non-human antibodies were completely replaced by the human form of the constant regions, and at least parts of the variable regions originating from non-human antibodies, excluding the three loops of amino acid sequences outside each variable region that bind to the target structure, were totally or partially replaced by corresponding parts of human antibodies.

[0042] The term Fab fragment refers to a region in an antibody that binds to antigens. The term humanized Fab fragment refers, also in analogy with the definition of the term humanized antibody above, to any Fab fragment in which the constant regions originating from non-human antibodies have been completely replaced by the human form of the constant regions and at least parts of the variable regions originating from non-human antibodies, excluding the three loops of amino acid sequences outside each variable region that bind to the target structure, have been totally or partially replaced by corresponding parts of human antibodies.

[0043] The term “peptide” refers to any peptide comprising one or more amino acid sequences of complementarity-determining regions (CDRs) defined in this application and which peptide is capable of binding to at least one epitope of human CLEVER-1. Preferred options

[0044] One embodiment of the present invention is directed to an agent capable of binding to human CLEVER-1, recognizing a CLEVER-1 epitope, wherein the epitope is discontinuous and comprises the amino acid sequences: PFTVLVPSVSSFSSR (SEQ ID NO: 1) and QEITVTFNQFTK (SEQ ID NO: 2) from human CLEVER-1,

[0045] and said agent comprises one or more sequences of Petition 870250108080, dated 11 / 26 / 2025, p. 15 / 116 10 / 50 amino acids from complementarity-determining regions (CDRs) that bind to so-called epitope sequences selected from the group consisting of TSGMGIG (SEQ ID NO: 7), HIWWDDDKRYNPALKS (SEQ ID NO: 8), HYGYDPYYAMDY (SEQ ID NO: 9), TASSSVSSSYLH (SEQ ID NO: 10), RTSNLAS (SEQ ID NO: 11) and HQYHRSPPT (SEQ ID NO: 12).

[0046] In some preferred embodiments of the present invention, the discontinuous epitope of human CLEVER-1 further comprises one or more amino acid sequences selected from the group consisting of ATQTGRVFLQ (SEQ ID NO: 3), DSLRDGRLIYLF (SEQ ID NO: 4), SKGRILTMANQVL (SEQ ID NO: 5) and LCVYQKPGQAFCTCR (SEQ ID NO: 6).

[0047] A part of the human CLEVER-1 target protein, for example, human stabilin-1, defined in SEQ ID NO: 31. The epitopes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in CLEVER-1 correspond to amino acids 420 to 434, 473 to 484, 390 to 399, 576 to 587, 615 to 627 and 313 to 327 of the human CLEVER-1 target protein defined in SEQ ID NO: 31.

[0048] In some preferred embodiments of the present invention, the agent capable of binding to a human CLEVER-1 epitope comprises at least two, preferably three, more preferably four, even more preferably five, and most preferably all six amino acid sequences of complementarity-determining regions (CDRs) defined above.

[0049] According to the present invention, the agent capable of Petition 870250108080, dated 11 / 26 / 2025, p. 16 / 116 11 / 50 ligand to human CLEVER-1 can be selected from the group consisting of an antibody, single-chain Fv or Fab fragment(s), peptide(s), or macromolecule(s).

[0050] In some preferred embodiments of the present invention, an agent capable of binding to human CLEVER-1 is a humanized antibody or a single-chain Fv or Fab fragment, and said humanized antibody or single-chain Fv or Fab fragment comprises a) constant regions of the human IgG heavy chain and kappa light chain, and b) one or more of the following sequences of complementarity-determining regions (CDRs)

[0051] i) of the heavy chain CDR 1: TSGMGIG (SEQ ID NO: 7) and / or CDR 2: HIWWDDDKRYNPALKS (SEQ ID NO: 8) and / or CDR 3: HYGYDPYYAMDY (SEQ ID NO: 9) and

[0052] ii) of the light chain CDR 1: TASSSVSSSYLH (SEQ ID NO: 10) and / or CDR 2: RTSNLAS (SEQ ID NO: 11) and / or CDR 3: HQYHRSPPT (SEQ ID NO: 12).

[0053] The humanized antibody or single-chain Fv or Fab fragment capable of binding to a human CLEVER-1 epitope by recognizing the discontinuous epitope sequences as defined above. The human CLEVER-1 discontinuous epitope comprises at least the sequences SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the human CLEVER-1 discontinuous epitope further comprises one or more sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0054] In some embodiments of the present invention referred to Petition 870250108080, dated 11 / 26 / 2025, p. 17 / 116 12 / 50 above, at least two, preferably three, more preferably four, even more preferably five, and most preferably all six CDRs defined above are included in the humanized antibody or single-chain Fv or Fab fragment.

[0055] In some embodiments of the present invention, the sequence of the variable region of the human IgG heavy chain of the humanized antibody or of the single-chain Fv or Fab fragment selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, preferably SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20. In some embodiments of the present invention, the sequence of the variable region of the human IgG light chain of the humanized antibody or of the single-chain Fv or Fab fragment selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30, preferably SEQ ID NO: 30.

[0056] In many embodiments of the humanized antibody or single-chain Fv or Fab fragment according to the invention, the constant regions of the human IgG heavy chain and kappa light chain are of this form. Human IgG4 constant regions are preferred. Many preferred embodiments comprise the human IgG4 light chain and IgG4 kappa with the L248E and / or, preferably, S241P mutations.

[0057] In some embodiments of the present invention, the humanized antibody or the single-chain Fv or Fab fragment is capable of binding to CLEVER-1 with a relative IC50 < 1.0, preferably < 0.8, more preferably < 0.6 and most preferably < 0.5 compared to the IC50 of monoclonal antibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 21, 2001).

[0058] According to one embodiment of the invention, the combination Petition 870250108080, dated 11 / 26 / 2025, p. 18 / 116 13 / 50 of the variable regions of the heavy and light chains of human IgG are selected from the combinations presented in Table 5 having binding capacity to human CLEVER-1 with a relative IC50 <1.0 compared to the IC50 of monoclonal antibody 3-372 (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 21, 2001).

[0059] A pharmaceutical composition, according to the invention, comprises the agent capable of binding to human CLEVER-1 or to the humanized antibody or to the single-chain Fv or Fab fragment described above and a suitable excipient. A modulation of tumor-promoting macrophages (M2) into pro-inflammatory macrophages (M1)

[0060] It was also discovered that an agent capable of binding to Human CLEVER-1, specifically specific epitope sequences in CLEVER-1 defined in the present application, can be used to activate macrophages to change their phenotype from M2 macrophages to M1 macrophages. Specifically, an agent capable of binding to CLEVER-1 in TAMs can be used to achieve a modulation of tumor-promoting macrophages (M2) into pro-inflammatory macrophages (M1). This modulation increases T cell activation and eventually leads, for example, to the removal of cancer-related immunosuppression. More precisely, it has been found that an agent capable of binding to specific sequences in the CLEVER-1 molecule can be used to remove immunosuppression by modulating M2 macrophages into M1 macrophages. Consequently, the present discovery provides a method to affect the immune system in an individual and is especially useful in cancer treatment or metastasis prevention, but is not limited to this approach.

[0061] Macrophages can be divided into two distinct phenotypes: M1 and M2 macrophages. M1 macrophages are proactive macrophages. Petition 870250108080, dated 11 / 26 / 2025, page 19 / 116 14 / 50 classic inflammatory cells, which produce large amounts of pro-inflammatory cytokines and co-stimulatory molecules, and are very efficient at activating T cell responses. M2 macrophages, in contrast, are immunosuppressive cells that synthesize anti-inflammatory cytokines and induce regulatory T cells, thus profoundly minimizing the activation of antigen-directed T cells. Tumor-associated macrophages (TAMs) are considered harmful as they mature into M2 macrophages (tumor-promoting macrophages) within the tumor environment and suppress the antitumor immune response and mediate angiogenic exchange, a crucial step in cancer growth. M2 macrophages can be modulated into M1 macrophages (pro-inflammatory macrophages), and such phenotypic conversion from M2 to M1 can directly or indirectly cause tumor rejection.

[0062] In the present context, the term “M1 macrophages” or “pro-inflammatory macrophages” refers to macrophages characterized by an increased measured level of macrophage / monocyte TNF-alpha (TNF-α) secretion or HLA-DR expression. Modulation of M2 macrophages into M1 macrophages will increase monocyte TNF-alpha secretion and also HLA-DR expression compared to control values ​​measured before administration of an agent capable of binding to human CLEVER-1 or the values ​​from one or more previous measurements taken at different times in the same patient. It is important to compare the measured values ​​of monocyte TNF-alpha secretion and HLA-DR expression with the values ​​of the same patient, since the level of these markers can vary from one individual to another and, for example, cytokines such as interferon-gamma and LPS activation can increase TNF-alpha expression by M2 macrophages.

[0063] It was surprisingly discovered that M2 macrophages Petition 870250108080, dated 11 / 26 / 2025, p. 20 / 116 15 / 50 can be activated to modulate M1 macrophages by contact of said macrophages with an agent capable of binding to human CLEVER-1, for example, by an antibody or a fragment thereof, peptide(s) or macromolecule(s) as defined in the present application. It has been found in particular that M2 macrophages associated with malignant tumors can be modulated or repolarized into M1 macrophages through contact of said macrophages with an agent capable of binding to human CLEVER-1 in TAMs. Both phenotypes can be present simultaneously and both phenotypes can be found in tumors.

[0064] An agent capable of binding to human CLEVER-1, such as an antigen or a fragment thereof, peptide(s) or macromolecule(s), is bound to human CLEVER-1 to achieve said modulation or repolarization of macrophage phenotypes. It has been identified that agents specific to the CLEVER-1 protein recognize a specific CLEVER-1 epitope sequence defined in the present application.

[0065] Specific binding to two or more of said epitope sequences in CLEVER-1 on TAMs will provide a novel method for treating cancers or preventing metastases without harmful side effects since treatment can be targeted to specific epitopes to achieve the desired modulation of macrophage phenotype. Consequently, the results described in this application are especially useful in the treatment or prevention of all types of malignant tumors associated with an increased number of tumor-promoting macrophages or other pathologies, such as chronic inflammation, in which an individual exhibits immunosuppressive dominance. Consequently, a method for treating cancer or preventing metastases comprising administering to an individual an agent capable of binding to human CLEVER-1, Petition 870250108080, dated 11 / 26 / 2025, page 21 / 116 16 / 50 preferentially targeting specific epitopes in the CLEVER-1 molecule defined above. The method comprises treatment or prevention of cancer by reducing tumor size and / or reducing tumor growth in an individual; and / or inhibiting the transmigration of cancer cells and the formation of metastases. In this way, any benign or malignant tumor or metastasis of a malignant tumor, such as skin cancer and colon cancer, can be treated. Leukemias, lymphomas, and multiple myelomas can also be treated. In particular, melanomas and lymphomas should respond very well to treatment based on animal models.

[0066] Macrophages also play an important role during inflammation and infection resolution, as well as affecting tumor growth or regression. In infections, a change from M1 to M2 macrophage can occur, leading to the generation of a suppressive environment that abolishes effector immunity. Consequently, the results described herein for modulating macrophage phenotype are also useful in the treatment of chronic infections to remove immunosuppression against infectious antigens. The invention also relates to a method for treating chronic infections comprising administering to an individual an agent capable of binding to CLEVER-1, preferably to two or more specific epitope sequences in the CLEVER-1 molecule defined in the present application, wherein said agent can activate macrophages to change their phenotype from M2 to M1.

[0067] In addition, an agent capable of binding to the molecule of CLEVER-1 in macrophages and monocytes in an individual can be used as an adjuvant in vaccines. This agent achieves repolarization of macrophages and, in this way, removes or at least reduces immunosuppression against vaccine antigens. Any antigen-induced vaccination can benefit if the site of Petition 870250108080, dated 11 / 26 / 2025, page 22 / 116 17 / 50 host or vaccination may be temporarily removed from immunosuppressive elements.

[0068] The modulation of M2 macrophages to M1 can be verified by measuring the secretion of TNF-alpha from monocytes from human blood samples. Consequently, increased TNF-alpha secretion can be used as a marker to monitor treatment response in an individual. TNF-alpha secretion can be determined from enriched peripheral blood monocytes collected from a patient's blood. A measured TNF-alpha level can be used as a marker for the patient's response to treatment comprising administration of an agent capable of binding to CLEVER-1 in the patient, when the level is compared to the control level measured in the same patient before administering said agent to the patient, or the values ​​of one or more previous measurements performed at different times in the same patient.

[0069] A method for estimating the efficiency of antiCLEVER-1 therapy by monitoring the development of M2 macrophage modulation into M1 macrophages when an agent capable of binding to CLEVER-1, preferably to said two or more CLEVER-1 specific epitope sequences, is administered to a patient, comprising the steps of (a) obtaining peripheral blood monocytes (PBLs) from a blood sample taken from said patient, (b) measuring TNF-α secretion from said PBLs and / or (c) measuring HLA-DR expression in CD14-positive PBLs and (d) compare the values ​​of TNF-α secretion and / or HLA-DR expression measured in steps (b) and (c) to control values ​​for an estimate of the effectiveness of anti-CLEVER-1 treatment, wherein the control values ​​are the values ​​measured before administration of a Petition 870250108080, dated 11 / 26 / 2025, page 23 / 116 18 / 50 agent capable of binding to CLEVER-1 in the patient or the values ​​of one or more previous measurements performed at different times in the same patient and in which an increase in TNF-alpha secretion or HLA-DR expression is indicative of the modulation of M2 macrophages into M1 macrophages.

[0070] The determination of TNF-alpha secretion from peripheral blood monocytes obtained from a blood sample taken from the patient can be performed using commonly known methods, for example, using a commercial TNF-alpha ELISA kit. HLA-DR expression in CD14-positive monocytes can also be monitored using a method known as flow cytometry.

[0071] The development of M2 macrophage modulation into M1 macrophages can be monitored by comparing a measured level of TNF-alpha secretion to control values ​​measured before administering an agent capable of binding to CLEVER-1 in the patient, or the values ​​of one or more previous measurements performed at different times in the same patient. For example, a reduced level of monocyte TNF-alpha secretion compared to previous measurement results or to a control can be used to indicate higher M2 macrophage expression, while an increased level of TNF-alpha compared to previous measurement results or to a control can be used to indicate higher M1 macrophage expression with lower M2 macrophage expression, which can also be used to indicate the effectiveness of antiCLEVER-1 treatment.The increased level of TNF-alpha indicates greater expression of M1 macrophages with less expression of M2 macrophages, that is, it attributes responsiveness to said therapy. An agent capable of binding to CLEVER-1 will activate at least a portion of the M2 macrophages to repolarize into M1 macrophages after administration of said agent. Petition 870250108080, dated 11 / 26 / 2025, page 24 / 116 19 / 50 Both macrophage phenotypes may be present, but increased expression of M1 macrophages may be observed compared to the situation before administration of said agent. Typically, at least a two-fold increase in measured TNF-alpha secretion compared to the control value is indicative of the modulation of M2 macrophages into M1 macrophages and thus indicates the patient's responsiveness to therapy. Diseases that respond to treatment

[0072] The balance of immune activation and suppression is very critical for the homeostasis of a human (or animal) in combating foreign material born from or entering the human (or animal). The example of Palani et al. (2016) is a physiological example of this and shows how local immunosuppression is fundamental to the well-being of an embryo in an environment dominated by the mother's immune defense. The same can occur in chronic infections, as some pathogens (e.g., tuberculosis) have learned to use a similar concealment mechanism against the host's immune system and could establish themselves in chronically infected sites (hepatitis). Eliminating this local immunosuppression could help the host fight these infections, as it would improve vaccination against these resistant pathogens.

[0073] Tumors have also adapted this immunosuppression to their advantage. The method, according to the present invention, for treating or preventing cancer by reducing the size of the malignant tumor; reducing malignant tumor growth; and / or inhibiting the transmigration of cancer cells and the formation of metastases is applicable to all forms of cancer. In this way, any benign or malignant tumor or metastasis of a malignant tumor, such as skin cancer and colon cancer, can be treated. Leukemias, lymphomas, and multiple myelomas can also be treated. In particular, melanomas and lymphomas should Petition 870250108080, dated 11 / 26 / 2025, page 25 / 116 20 / 50 responded very well to treatment based on animal models.

[0074] It is believed that the agent capable of binding to CLEVER-1 or a humanized antibody or single-chain Fv or Fab fragment according to the present invention or the pharmaceutical composition according to the present invention is useful in the treatment or prevention of all types of sarcomas, for example, fibrosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, and rhabdomyosarcoma, mesothelioma, meningoma, leukemias, lymphomas, as well as all types of carcinomas, such as squamous cell carcinomas, basal cell carcinomas, adenocarcinomas, papillary carcinomas, cystadenocarcinomas, bronchogenic carcinomas, melanomas, renal cell carcinomas, hepatocellular carcinoma, transitional cell carcinomas, choriocarcinomas, seminomas and embryonal carcinomas.

[0075] An agent capable of binding to human CLEVER-1 on an individual or humanized antibody or single-chain Fv or Fab fragment or a pharmaceutical composition according to the invention suitable for use in removing tumor or antigen-induced immunosuppression by modulating M2 macrophages into M1 macrophages, wherein the agent binds to a human CLEVER-1 epitope sequence defined in the present application.

[0076] An agent capable of binding to human CLEVER-1 in an individual or humanized antibody or single-chain Fv or Fab fragment or pharmaceutical composition, according to the invention, is suitable for use in the treatment or prevention of cancer by reducing the size of malignant tumors; reducing malignant tumor growth in an individual; and / or inhibiting cancer cell transmigration and metastasis formation, wherein immunosuppression around malignant growth is removed through modulation of M2 macrophages into M1 macrophages. Petition 870250108080, dated 11 / 26 / 2025, page 26 / 116 21 / 50

[0077] An agent capable of binding to human CLEVER-1 in an individual or humanized antibody or single-chain Fv or Fab fragment or pharmaceutical composition, according to the invention, is also suitable for use in the treatment of chronic infections in an individual, wherein immunosuppression against infectious antigens is removed by modulating M2 macrophages into M1 macrophages.

[0078] An agent capable of binding to human CLEVER-1 in an individual or humanized antibody or single-chain Fv or Fab fragment or pharmaceutical composition, according to the invention, is also suitable for use as a vaccine adjuvant, wherein immunosuppression against vaccine antigens is removed by modulating M2 macrophages into M1 macrophages.

[0079] A method for modulating M2 macrophages into M1 macrophages comprises administering to a subject in need thereof, an agent capable of binding to CLEVER-1, preferably capable of binding to specific sequences on the CLEVER-1 molecule, as defined in the present application. Said method can be used in the treatment of cancer or in the prevention of metastases in an individual or in the treatment of chronic infections in an individual. The response to treatment can be verified by measuring the TNFα secretion of said PBLs and / or the HLA-DR expression in CD14-positive PBLs, as described in the present application. Routes of administration, formulations and required dosage.

[0080] The pharmaceutical compositions to be used in the present invention may be administered by any means that achieves their intended purpose. For example, administration may be intravenous, intra-articular, intratumoral, or subcutaneous. In addition to the pharmacologically active compounds, the pharmaceutical preparations of the compounds preferably contain suitable vehicles. Petition 870250108080, dated 11 / 26 / 2025, page 27 / 116 22 / 50 pharmaceutically acceptable comprising excipients and auxiliaries that facilitate the processing of the active compounds into preparations that can be used pharmaceutically.

[0081] The chosen dose must be therapeutically effective in relation to the disease being treated. Therefore, immunosuppression must be sufficient to treat the disease without effects that essentially jeopardize the desired outcome of the treatment. When treating or preventing cancer, the dose must be sufficient to reduce the size of the malignant tumor, reduce malignant tumor growth, and / or inhibit the transmigration of cancer cells and the formation of metastases. The dose is dependent on the turnover of the administered agent, but normally these treatments follow a regimen of 1 to 5 mg / kg every 2 to 4 weeks. EXAMPLES

[0082] The experimental section that follows illustrates the invention by providing examples.

[0083] Examples 1 to 10 illustrate the discontinuous epitope mapping of human CLEVER-1 and the generation of humanized antibodies from mouse monoclonal antibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 21, 2001) using Composite Human Antibody™ technology. The ability of anti-CLEVER-1 antibodies to promote immune activation is illustrated in Examples 11 to 14.

[0084] Example 1 illustrates the complete discontinuous epitope mapping of antibodies targeting human CLEVER-1.

[0085] Examples 2 to 6 illustrate the determination of the heavy and light chain (VH and VK) region sequences of clone 3-372 of the anti-Clever 1 antibody and the production of chimeric antibodies comprising the variable regions of 3-372 and constant regions of the kappa light chain and Petition 870250108080, dated 11 / 26 / 2025, page 28 / 116 23 / 50 of the human IgG4 heavy chain. mRNA was extracted from hybridoma clone 3-372, reverse transcribed, amplified by PCR, and antibody-specific transcripts were cloned. The nucleotide and amino acid sequences of the variable regions of the antibody heavy and light chains were determined, and sequence data analysis for humanization was performed using Antitope's proprietary Composite Human Antibody™ technology.

[0086] Examples 2 to 6 demonstrate that: The variable regions of the mouse anti-Clever 1 antibody 3-372 were cloned and sequenced. The genes from the variable region were combined with the constant regions of the heavy chain and kappa light chain of human IgG4(S241P) and expressed in NSO cells to produce a chimeric anti-Clever 1 antibody. A competitive ELISA assay from the NSO-derived chimeric antibody was used to demonstrate that the binding efficiency of the chimeric antibody to Clever 1 is similar to that of the parental murine antibody.

[0087] Examples 7 to 10 illustrate: Design of composite human anti-CLEVER-1 antibodies that were expressed and tested for binding to human Clever-1. The key residues involved in the structure and binding of anti-CLEVER-1 were determined by structure-homology modeling to generate a “residue restriction map”. The residue restriction map was used as a template to source human V-region sequence segments from databases containing unrelated human antibody sequences. Each selected sequence segment, as well as the junctions between segments, was tested for the presence of potential T-cell epitopes using in silico analyses (iTope™ and TCED™). Using this method, all variants of the Composite Human Antibody™ sequence were designed to avoid T-cell epitopes. The genes of the Composite Human Antibody™ V-region Petition 870250108080, dated 11 / 26 / 2025, page 29 / 116 24 / 50 antibodies were generated using synthetic oligonucleotides encoding combinations of selected human sequence segments. These were then cloned into vectors containing constant regions of the heavy chain and kappa light chain of human IgG4(S241P), and the antibodies were produced and tested for binding to the target antigen by competitive ELISA compared to the original murine monoclonal reference antibody.

[0088] Examples 7 to 10 demonstrate the construction of four VH and five Vk regions of the Composite Human Antibody™. Combinations of heavy and light composite chains were expressed in NS0 cells, purified, and tested for CLEVER-1 binding in a competitive ELISA assay. The results demonstrated that the binding efficiency of many of the Composite Human Antibodies™ to CLEVER-1 was at least as good as that of the chimeric reference antibody, and several were markedly better. Based on the absence of a potential glycosylation site and an unpaired cysteine, and on generated expression and binding efficiency datasets, three potential indications were designated as follows: VH2 / VK5, VH3 / VK5, and VH4 / VK5.

[0089] Examples 11 and 12 illustrate the binding of anti-CLEVER-1 antibody to human peripheral blood monocytes and the activation of TNF-alpha secretion in human peripheral blood monocytes. Example 13 illustrates the mode of action of anti-CLEVER-1 antibodies on tumor-associated macrophages in syngeneic mouse cancer models.

[0090] Example 14 illustrates that the binding of CLEVER-1 to antibodies 9-11 and 3-372 promotes opposite effects on mTOR and c-Jun signaling in human peripheral blood monocytes. Example 1 Complete mapping of discontinuous epitopes of CLEVER-1 Petition 870250108080, dated 11 / 26 / 2025, page 30 / 116 25 / 50 human

[0091] Experimental discontinuous epitopes for four antibodies were established using Pepscan analysis. Antibodies FAR02 VH3 / VK5 and FU-HI-3-372 target the same discontinuous epitope, while antibodies 3-266 and AK FUMM 9-11 target other distinct epitopes. The study was conducted at Pepscan Presto BV, (Zuidersluisweg 2, 8243RC Lelystad, Netherlands).

[0092] Antibodies 3-266, FAR02 VH3 / VK5, FU-HI-3-372 and AK FUMM 9-11 were supplied by Faron Pharmaceuticals Oy.

[0093] The human CLEVER-1 target protein, for example, human stabilin1, was defined by SEQ ID NO: 31. Disulfide bridges connect residues (Uniprot numbering STAB1_HUMAN): 112 126 | 120 136 | 138 147 | 160 171 | 164 181 | 183 192 199 210 | 204 217 | 236 247 | 241 257 | 259 270 | 732746 740 756 | 758 767 | 822 837 | 831 846 | 865 879 | 873 889 891 902 | 908 922 | 916 932 | 934 945 | 951 964 | 958 974 CLIPS Technology

[0094] The CLIPS technology employed structurally fixes peptides into defined three-dimensional structures. This results in functional mimetics of even the most complex binding sites. CLIPS technology is now routinely used to shape peptide libraries into single, double, or triple loop structures, as well as sheet and helix folds. The CLIPS reaction occurs between the bromine groups of the CLIPS structure and the thiol side chains of the cysteines. The reaction is fast and specific under mild conditions. Using this elegant chemistry, native protein sequences are transformed into CLIPS constructs with a range of structures. (Timmerman et al., J. Mol. Recognit. 2007; 20: 283-29)

[0095] CLIPS library screening begins with converting the target protein into a library of up to 10,000 constructs Petition 870250108080, dated 11 / 26 / 2025, page 31 / 116 26 / 50 overlapping peptides, using a combinatorial array design. In a solid carrier, a matrix of linear peptides is synthesized, which are subsequently molded into spatially defined CLIPS constructs. Constructs representing both parts of the discontinuous epitope in the correct conformation bind to the antibody with high affinity, which is detected and quantified. Constructs presenting the incomplete epitope bind to the antibody with lower affinity, while constructs that do not contain the epitope do not bind. Affinity information is used in iterative screenings to define the sequence and conformation of epitopes in detail. The target protein containing a discontinuous conformational epitope is converted into an array library. Combinatorial peptides are synthesized on a proprietary minicard and chemically converted into spatially defined CLIPS constructs. Heat map analysis

[0096] A heat map is a graphical representation of data in which the values ​​taken by a variable on a two-dimensional map are represented as colors.

[0097] For double-loop CLIPS peptides, such a two-dimensional map can be derived from the independent sequences of the first and second loops. For example, the sequences of the 16 CLIPS peptides are effective permutations of, for example, 4 unique subsequences, for example, in loop 1 and 4 unique subsequences, for example, in loop 2. In this way, the observed ELISA data can be plotted on a 4x4 matrix, where each X coordinate corresponds to the sequence of the first loop, and each Y coordinate corresponds to the sequence of the second loop.

[0098] To make visualization even easier, the ELISA values ​​can be replaced with colors from a continuous gradient. By Petition 870250108080, dated 11 / 26 / 2025, p. 32 / 116 27 / 50 example, extremely low values ​​can be colored green, extremely high values ​​colored red, and average values ​​colored black. Peptide synthesis

[0099] To reconstruct epitopes of the target molecule, a peptide library was synthesized. An amino-functionalized polypropylene scaffold was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) and subsequent cleavage of the Boc groups using trifluoroacetic acid (TFA). Standard Fmoc peptide synthesis was used to synthesize peptides on the amino-functionalized solid scaffold, using custom-modified JANUS liquid handling stations (Perkin Elmer).

[00100] The synthesis of structural mimetics was performed using Pepscan's proprietary Chemically Linked Peptides in Structures (CLIPS) technology. CLIPS technology allows peptides to be structured into single loops, double loops, triple loops, sheet folds, helix folds, and combinations thereof. CLIPS templates are coupled to cysteine ​​residues. The side chains of multiple cysteines in the peptides are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of CLIPS P2 (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8) / acetonitrile (1:3 (v / v)). This solution is added to the peptide matrices. The CLIPS model will bind to two cysteine ​​side chains, as present in solid-phase linked peptides from peptide clusters (455-well plate with 3 μL wells).The peptide matrices are gently stirred in the solution for 30 to 60 minutes until they are completely submerged. Petition 870250108080, dated 11 / 26 / 2025, page 33 / 116 28 / 50 Finally, the peptide matrices are washed extensively with excess H2O and sonicated in disruption buffer containing 1% SDS / 0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70 °C for 30 minutes, followed by sonication in H2O for a further 45 minutes. The CLIPS T3 carrier peptides were made in a similar manner, but now with three cysteines. ELISA screening

[00101] Antibody binding to each of the synthesized peptides was tested using a PEPSCAN-based ELISA. Peptide matrices were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide matrices were incubated with a 1 / 1000 dilution of an appropriate antibody peroxidase conjugate (SBA; Table 1) for one hour at 25°C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzothiazoline sulfonate (ABTS) and 20 μL / mL of 3 percent H2O2 were added. After one hour, color development was measured. Color development was quantified using a charge-coupled device (CCD) camera and an image processing system. Table 1 Antibody details Supplier Name | Cat. No. | HRP-conjugated goat anti-human antibody | Southern Biotech | 2010-05 | HRP-conjugated rabbit anti-mouse IgG(J+L) antibody | Southern Biotech | 6175-05 | HRP-conjugated goat anti-mouse IgG(H+L) antibody | Southern Biotech | 3010-05

[00102] Langedijk et al. (2011). Helical peptide matrices for principal identification and interaction site mapping, Analytical Biochemistry 417: 149-155 Peptide project Petition 870250108080, dated 11 / 26 / 2025, page 34 / 116 29 / 50

[00103] Different sets of peptides were synthesized according to the following designs. Note that in some sets the peptides were synthesized in a random order. Below, the actual order of the peptides is shown. Set 1 Mimicry Type Linear Marker LIN Description 15-mer linear peptides derived from the human Clever-1 target sequence with a one-residue displacement. Sequences (first 10) QVLFKGCDVKTTFVT VLFKGCDVKTTFVTH LFKGCDVKTTFVTHV FKGCDVKTTFVTHVP KGCDVKTTFVTHVPC GCDVKTTFVTHVPCT CDVKTTFVTHVPCTS DVKTTFVTHVPCTSC VKTTFVTHVPCTSCA KTTFVTHVPCTSCAA Set 2 Mimicry type: Linear marker LIN.AA. Description: Peptides from set 1, but with residues at positions 10 and 11 substituted by Ala. Since a native Ala occurs at any position, it is substituted by Gly. Sequences (first 10): GAETPCNGHAACLDG LTMANQVLAAAISEE ILLPPTILPAAPKHC DRNGTCVCQAAFRGS PGYTQQGSEAAAPNP Petition 870250108080, dated 11 / 26 / 2025, page 35 / 116 30 / 50 PIDPCRAGNAACHGL HTDALCSYVAAGQSR KGCDVKTTFAAHVPC CQALNTSTCAANSVK RAVGGGQRVAACPPG Set 3 Linear mimicry type. Marker LIN20.C. Description: Linear peptides of length 20 are derived from the human Clever-1 target sequence with a one-residue displacement. Cys residues are protected by acetimidomethyl (Acm, denoted “2”). Sequences (first 10): 2H2PENYHGDGMV2LPKDP2 SGWLRELPDQITQD2RYEVQ LAQH2HLHAR2VSQEGVAR2 IKKQT2PSGWLRELPDQITQ 2RESEVGDGRA2YGHLLHEV QRV2T2PPGFGGDGFS2YGD NGVFHVVTGLRWQAPSGTPG AT2QVTADGKTS2V2RESEV KYSYKYKDQPQQTFNIYKAN 2VYIHDPTGLNVLKKG2ASY Set 4 Linear mimicry type. Marker LIN25.C. Description: Linear peptides of length 25 are derived from the human Clever-1 target sequence with a one-residue shift. Cys residues are protected by Acm (“2”). Sequences (first 10): KKG2ASY2NQTIMEQG22KGFFGPD PD2QSV2S2VHGV2NHGPRGDGS2L GPGQSR2T2KLGFAGDGYQ2SPIDP IFPKE2VYIHDPTGLNVLKKG2ASY PTILPILPKH2SEEQHKIVAGS2VD Petition 870250108080, dated 11 / 26 / 2025, page 36 / 116 31 / 50 ENFRGSA2QE2QDPNRFGPD2QSV2 QNTQ2SAEAPS2 R2LPGYTQQGS E2 GRV2VAIDE2ELDMRGG2HTDAL2S APSGTPGDPKRTIGQILASTEAFSR DGMV2LPKDP2TDNLGG2PSNSTL2 Set 5 Type of mimicry: Restricted peptides, mP2 CLIPS. Marker: LOOP. Description: Peptides of length 17. At positions 2 to 16 are 15-mer sequences derived from the target protein. At positions 1 and 17, Cys residues are joined by mP2 CLIPS. Native Cys residues are protected by Acm (“2”). Sequences (first 10): CL2SYVGPGQSR2T2KC, C2SYVGPGQSR2T2KLC, CSYVGPGQSR2T2KLGC, CYVGPGQSR2T2KLGFC, CVGPGQSR2T2KLGFAC, CGPGQSR2T2KLGFAGC, CPGQSR2T2KLGFAGDC, CGQSR2T2KLGFAGDGC, CQSR2T2KLGFAGDGYC, CSR2T2KLGFAGDGYQC Set 6 Type of mimicry: Linear disulfide mimetics. Marker: CYS22. Description: Linear disulfide mimetics of length 22 designed based on Uniprot information on disulfide bridges for human CLEVER-1. Cys residues within a mimetic, which do not participate in disulfide bridge formation, are protected by Acm (“2”). Sequences (first 10) Petition 870250108080, dated 11 / 26 / 2025, page 37 / 116 32 / 50 WGSR2HECPGGAETP2NGHGTC SR2HECPGGAETP2NGHGTCLD 2HECPGGAETP2NGHGTCLDGM ECPGGAETP2NGHGTCLDGMDR GAETPCNGHGT2LDGMDRNGTC ETPCNGHGT2LDGMDRNGTCV2 PCNGHGT2LDGMDRNGTCV2QE LDGMDRNGT2VCQENFRGSACQ GMDRNGT2VCQENFRGSACQE2 DRNGT2VCQENFRGSACQE2QD Set 7 Mimicry Type Combinatorial Disulfide Bridge Mimetic Marker CYS27 Description Combinatorial peptides of length 27. At positions 1 to 11 and 16 to 27 are 11-mer sequences derived from the target sequence on page 7 joined by the “GGSGG” linker. This peptide set was designed based on disulfide bridge information obtained from Uniprot. Cys residues within a mimetic, which do not participate in the formation of the disulfide bridge, are protected by Acm (“2”). Sequences (top 10) PGYWGSR2HECGGSGGAETP2NGHGTC YWGSR2HECPGGGSGGAETP2NGHGTC GSR2HECPGGAGSGGAETP2NGHGTC R2HECPGGAETGGSGGAETP2NGHGTC HECPGGAETP2GGSGGAETP2NGHGTC CPGGAETP2NGGGSGGAETP2NGHGTC PGYWGSR2HECGGSGGTP2NGHGTCLD YWGSR2HECPGGGSGGTP2NGHGTCLD GSR2HECPGGAGGSGGTP2NGHGTCLD R2HECPGGAETGGSGGTP2NGHGTCLD Set 8 Type of mimicry: Discontinuous matrix, T3 CLIPS Petition 870250108080, dated 11 / 26 / 2025, page 38 / 116 33 / 50 MAT marker Description: Peptides of length 33. At positions 2 to 16 and 32 are 15-mer peptides derived from the human Clever-1 target sequence. At positions 1, 17, and 33, the Cys residues are joined by T3 CLIPS. The native Cys residues are protected by Acm (“2”). Sequences (first 10) CPNRFGPD2QSV2S2VCV2S2VHGV2NHGPRGC CHGDGMV2LPKDP2TDCSAG2FAF2SPFS2DRC C2VD2QALNTST2PPNCPKH2SEEQHKIVAGSC CPKH2SEEQHKIVAGSCGPD2TQ2PGGFSNP2C CRYEVQLGGSMVSMSGCVP2TS2AAIKKQT2PC CHKIVAGS2VD2QALNCIHMLDGILLPPTILPC CF2T2RPGLVSINSNACVTADGKTS2V2RESEC C2VYIHDPTGLNVLKKCGSGGV2QQGT2APGFC CLRVAVAMMDQG2REICDGRA2YGHLLHEVQKC CYSYKYKDQPQQTFNICHEVQKATQTGRVFLQC Tracking details

[00104] Antibody binding depends on a combination of factors, including antibody concentration and the amounts and nature of competing proteins in the ELISA buffer. Additionally, pre-coating conditions (the specific treatment of peptide matrices before incubation with the experimental sample) affect binding. These details are summarized in Table 2. For Pepscan buffer and pre-conditioning (SQ), the numbers indicate the relative amount of competing protein (a combination of horse serum and ovalbumin). Table 2 Tracking conditions Sample Buffer Dilution Marker Preconditioning 3-266 5 pg / mL 10%SQ 10%SQ AK FUMM 9-11 1 pg / mL 50%SQ 50%SQ Petition 870250108080, dated 11 / 26 / 2025, page 39 / 116 34 / 50 FAR02 VH3 / VK5 3 pg / mL 1%SQ 1%SQ FU-HI_3-372 5 pg / mL 1%SQ 1%SQ Results Antibody 3-266

[00105] When tested under high stringency conditions, antibody 3-266 did not bind to any peptides present in the arrays. When tested under low stringency conditions, the antibody bound to peptides from all sets. Results obtained with single epitope mimetics suggest that the sequence 1030QWLKSAGITLPADRR1044 represents the dominant part of the epitope. Data obtained with combinatorial epitope mimetics (Figure 1) suggest that the antibody also recognizes the sequence 857LHARCVSQEGVARCR871. In addition, a weak consistent signal was recorded for the peptide sequence with 435TMNASLAQQLCRQHI450. Figure 1b illustrates the heat map representation of the results obtained for antibody AK FUMM 3-266 in set 8 (discontinuous epitope mimetics). The average signal is drawn in black, and the extremely high signal is drawn in a lighter color. Enclosed regions are enlarged. AK FUMM 9-11 Antibody

[00106] When tested under high stringency conditions, the AK-FUMM 9-11 antibody bound to peptides from all sets. Results obtained with single epitope mimetics suggest that the 885PSNPCSHPDRGG896 antibody represents the dominant part of the epitope. Data obtained with combinatorial epitope mimetics suggest that the antibody also recognizes combinatorial epitope mimetics containing the sequence 166FRGSACQECQDPNRF180 (Figure 1b). Figure 1b illustrates the heat map representation of the results obtained for the AK-FUMM 9-11 antibody in set 8 (discontinuous epitope mimetics). The average signal is plotted in black. Petition 870250108080, dated 11 / 26 / 2025, page 40 / 116 35 / 50 and the extremely high signal is drawn in a lighter color. Enclosed regions are enlarged. Antibody FAR02 VH3 / VK5 and FU-H 1:3-372

[00107] When tested under high stringency conditions, the FAR02 VH3 / vk5 and FU- FU-HI-3-372 antibodies did not bind to any peptides present in the matrices. When tested under low stringency conditions, these antibodies specifically bind only to the peptides in set 8 (Figure 2). Analysis of the results obtained with the discontinuous mimic suggests that the antibody recognizes a discontinuous epitope composed of sequences 390ATQTGRVFLQ399 (SEQ ID NO: 3), 420PFTVLVPSVSSFSSR434 (SEQ ID NO: 1), 473QEITVTFNQFTK484 (SEQ ID NO: 2), 576DSLRDGRLIYLF587 (SEQ ID NO: 4), 615SKGRILTMANQVL627 (SEQ ID NO: 5), where sequences 473QEITVTFNQFTK484 (SEQ ID NO: 2) and 420PFTVLVPSVSSFSSR434 (SEQ ID NO: 1) appear to represent major epitopes. An additional weaker signal was recorded for discontinuous mimetics containing the sequence. 313LCVYQKPGQAFCTCR327 (SEQ ID NO: 6). The results obtained with simple epitope mimetics do not allow for epitope calling. Figure 2 illustrates the heat map representation of the results obtained for the FU-HI-3-372 antibody in set 8 (discontinuous epitope mimetics). The average signal is plotted in black and the extremely high signal is plotted in a lighter color. Boxed regions are enlarged. Conclusions

[00108] The antibodies were tested against Pepscan peptide matrices. It was possible to identify experimental discontinuous epitopes for all monoclonal antibodies. The peptide sequences comprising the epitopes are listed in Table 3. Antibodies 3-266 and AK FUMM 9-11 bind to distinct discontinuous epitopes. Petition 870250108080, dated 11 / 26 / 2025, page 41 / 116 36 / 50 The FAR02 VH3 / VK5 and FU-HI-3-372 antibodies exhibited highly similar binding patterns when tested in matrices and therefore showed recognition of the same discontinuous epitope in the FAS1 / FAS2 domains. Table 3. Epitopes found. Antibody Epitope Sequences Domain 3-266 1030QWLKSAGITLPADRR1044 857LHARCVSQEGVARCR871 435TMNASLAQQLCRQHI450 FAS 3 Type EGF-6 FAS 1 AK FUMM 9-11 885PSNPCSHPDRGG896 166FRGSACQECQDPNRF180 Type EGF-6 Type EGF-1 FAR02 VH3 / VK5 + FU- HI-3-372 420PFTVLVPSVSSFSSR434 473QEITVTFNQFTK484 390ATQTGRVFLQ399 576DSLRDGRLIYLF587 615SKGRILTMANQVL627 FAS 1 FAS 1 FAS 1 FAS 2 FAS 2

[00109] To compare experimentally identified epitopes for the aforementioned antibodies, a schematic drawing was used in Figure 3. This scheme was adapted from Figure 1 from Kzhyshkowska, The Scientific World JOURNAL (2010), representing 10 2039-2053 Stabilin-1 (smart domain organization-1). Figure 3 schematically illustrates the organization of the CLEVER-1 domain (aa_25-1027 according to the target sequence). The arrows indicate relative positions of identified binding motifs. The circular arrows indicate positions of dominant epitope cores. Example 2 mRNA extraction, RT-PCR and cloning

[00110] mRNA was successfully extracted from hybridoma cells (PolyA Tract system, Promega Cat. No. Z5400). RT-PCR was performed using degenerate primer sets for signal sequences. Petition 870250108080, dated 11 / 26 / 2025, page 42 / 116 37 / 50 murine specimens with a single constant region primer. The heavy chain variable region mRNA was amplified using a set of six degenerate primer clusters (HA to HF), and the light chain variable region mRNA was amplified using a set of eight degenerate primer clusters (kA to kG and AA). Amplification products were obtained with the heavy chain primer sets HD and the light chain primer sets kB, kC, and kG, confirming that the light chain is from cluster k (Figure 4). Each product was cloned, and multiple clones of each were sequenced.

[00111] Using this methodology, a single VH sequence [SEQ ID NO: 32 (base sequence) and NO: 33 (amino acid sequence)] was identified in the HD group and a single functional Vk sequence [SEQ ID NO: 34 (base sequence) and NO: 35 (amino acid sequence)] was identified in the kG primer group. Heavy chain CDRs extend from base 91 to 111 (SEQ ID NO: 7), 154 to 210 (SEQ ID NO: 8) and 298 to 333 (SEQ ID NO: 9); and light chain CDRs extend from base 70 to 105 (SEQ ID NO: 10), 151 to 171 (SEQ ID NO: 11) and 268 to 294 (SEQ ID NO: 12). The definitions of CDR and protein sequence numbering are in accordance with Kabat. An aberrant k-light chain transcript normally associated with the SP2 / 0 hybridoma fusion partner (GenBank M35669) was also identified in both kB and kC primer groups.

[00112] Figure 4 illustrates the separation on a 1% agarose gel of the RT-PCR products of hybridoma 3-372. The gel was stained with SYBR® Green dye (Invitrogen Cat. No. S-7567) and photographed under ultraviolet light. The size marker is GeneRuler™ 1Kb Plus (Fermentas Cat. No. SM1331). The boxes indicate bands that were isolated for cloning and sequencing. Example 3 Sequence analysis Petition 870250108080, dated 11 / 26 / 2025, p. 43 / 116 38 / 50

[00113] The analysis of the sequences obtained from the hybridoma expressing 3-372 is summarized in Table 4. Table 4 Sequence analysis of antibody 3-372a H Chain L Chain CDR 1 Length 7aa 12aa CDR 2 Length 16aa 7aa CDR 3 Length 12aa 9aa Closest Human Germline b IGHV2-5*10 (73%) IGKV3D-20*01 (65%) Closest Human Germline FW1b IGHV2-70*06 (73%) IGKV1D-17*01 (68%) Closest Human Germline FW2b IGHV2-5*09 (86%) IGKV1D-39*01 (73%) Closest Human Germline FW3b IGHV2-70*13 (72%) IGKV1D-43*01 (78%) Closest Human Germline Jb IGHJ1 (91%) IGKJ4 (80%) CDR definitions and sequence numbering according to the Kabat germline ID indicated followed by % homology

[00114] Structure and homology analysis of the murine 3-172 variable domain sequence identified four structural residues in the variable region of the heavy chain and five structural residues in the variable region of the light chain that were considered critical or possibly important for antigen binding (“restrictive residues”). Analysis of additional sequence databases revealed that segments of the human main chain can be found to include all Petition 870250108080, dated 11 / 26 / 2025, page 44 / 116 39 / 50 of the desirable restriction residues and all RDF residues, thus enabling the construction of Composite Human Antibodies™.

[00115] It was also observed that the V 3-372 regions contain some unusual features. The VH chain contains an N-glycosylation site at the beginning of CDR1 since residue 30 is N and 32 is S (the NXS or NXT N-glycosylation signal, where X can be any amino acid). Due to the likely exposure of this motif to the antibody surface, this site is likely to be glycosylated. Therefore, it would be advantageous (if not involved in antigen binding) to avoid this site in Composite Human Antibodies in order to avoid any production problems in the future. The Vk chain also contains a glycosylation site, but only in the context of a human constant k region, since the final amino acid of the Vk domain is asparagine. The mouse constant k domain initiates RA, while the human constant k domain initiates RT, thus forming a glycosylation signal. Therefore, sequences for Composite Human Antibodies™ will be selected to avoid this asparagine.

[00116] The k domain also contains an unpaired cysteine ​​at position 47. Molecular modeling suggests that this residue will be inserted into the structure and is therefore not available for disulfide linkage; however, it could be a key residue for maintaining the Vk conformation of CDR2 and therefore sequences for Composite Human Antibodies™ will be selected with and without this residue (the latter including the human consensus L at this position) in order to investigate its effects on antigen binding. Example 4 Chimeric antibody expression

[00117] Variable regions 3-372 were transferred to the Antitope expression vector system for the heavy chain and Petition 870250108080, dated 11 / 26 / 2025, page 45 / 116 40 / 50 kappa light chain of IgG4 (S241P). NSO cells were transfected via electroporation and selected using methotrexate (MTX). Several MTX-resistant colonies were identified using an ELISA for kappa chain detection / Fc capture, and IgG expression-positive cell lines were continuously expanded from 96-well plates to T175 flasks in medium containing gradually increasing concentrations of MTX and subsequently frozen under liquid nitrogen. At each stage, IgG expression was quantified.

[00118] Chimeric IgG4 3-372 was purified from cell culture supernatants on a Sepharose Protein A column and quantified by OD280 nm using an extinction coefficient (Ec (0.1%)) value of 1.55 based on the predicted amino acid sequence for chimeric IgG4. Approximately 90 μg of antibody were purified and a sample was analyzed by reduction with SDSPAGE (Figure 5).Bands corresponding to the predicted sizes of the heavy and light chains were observed without evidence of any contamination; however, it was noteworthy that the chimeric light chain appears to be glycosylated, as evidenced by its higher apparent molecular weight compared to the mouse light chain. The heavy chain also appeared to be running slower than normal, suggesting that it is also N-glycosylated; however, digestion with glycosidases would be necessary to demonstrate that this is indeed the case.

[00119] Figure 5 illustrates the SDS-PAGE gel stained with Coomassie blue of chimeric IgG4 3-372 purified in protein A. 1 μg of sample was loaded onto a NuPage 4-12% Bis-Tris gel (Invitrogen Cat. No. NP0322BOX) and run at 200 V for 30 min. Columns 1 and 4: Pre-labeled protein standard (Fermentas PageRuler Cat. No. SM1811). Column 2: 1.0 pg of chimeric IgG4 antibody 3-372. Column 3: 1.0 pg of murine antibody 3-372. Petition 870250108080, dated 11 / 26 / 2025, p. 46 / 116 41 / 50 Example 5 Binding of the chimeric antibody to CLEVER-1

[00120] The chimeric 3-372 binding of NS0 to CLEVER-1 was evaluated by competitive ELISA. Briefly, a 96-well Maxisorp Nunc Immulon plate (Fisher Cat. No. DIS-971-030J) was coated with CLEVER-1 at 1 pg / mL in PBS (100 μL / well) overnight at 4 °C, with an additional 1 hour at 37 °C the following morning. The wells were washed with PBS / Tween 20 0.1% and then blocked for 45 min at room temperature in Marvel 1% / BSA 1% / PBS.

[00121] Both dilution series of Chimeric 3-372 and reference mouse 3-372 (5-0.078 pg / mL) were premixed with a constant concentration (0.6 pg / mL) of biotinylated mouse 3-372 antibodies in 2% BSA / PBS. The blocked ELISA plate was washed as before and 100 μL of the premixed antibodies were added to each well. The plate was incubated for 1 hour at room temperature. Binding of biotinylated mouse 3-372 to CLEVER-1 was detected using Streptavidin-HRP (Sigma Cat. No. S5512) and TMB single solution substrate (Invitrogen Cat. No. 00-2023). The reaction was stopped with 3M HCl, the absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader, and the binding curve of the chimeric 3-372 was compared with that of the reference mouse 3-372 antibody (Figure 6).

[00122] Figure 6 shows the binding profile of mouse and chimeric antibodies to CLEVER-1 in competition with biotinylated mouse antibody. The curves are nearly identical, giving IC50 values ​​of 0.89 pg / mL for the chimeric antibody compared to 0.77 pg / mL for the mouse antibody. This confirms that the correct variable region sequences were identified and expressed in the chimeric antibody. Example 7 Petition 870250108080, dated 11 / 26 / 2025, page 47 / 116 42 / 50 Project on Sequences and Variable Region Variants of Composite Human Antibody™

[00123] Structural models of the V regions of the murine antibody antiCLEVER-1 were produced using the Swiss PDB and analyzed in order to identify important “restriction” amino acids in the V regions that were likely essential for the antibody’s binding properties. The residues contained in the CDRs (using both Kabat and Chothia definitions) along with several structural residues were considered important. Both the VH and Vk sequences of anti-Clever 1 contain typical structural residues, and the motifs of CDRs 1, 2, and 3 are comparable to many murine antibodies. However, a potential site for N-linked glycosylation was identified in the VH sequence (30N) and an unpaired cysteine ​​in the Vk sequence (47C).

[00124] From the above analysis, it was considered that human composite anti-CLEVER-1 sequences could be created with a wide latitude of alternatives outside the CDRs, but with only a restricted menu of possible alternative residues within the CDR sequences. Preliminary analysis indicated that corresponding sequence segments from various human antibodies could be combined to create CDRs similar or identical to those of murine sequences. For CDRs, a wide selection of human sequence segments was identified as possible components of the new V Composite Human Antibody™ regions.

[00125] Based on the above analysis, a large preliminary set of sequence segments that could be used to create variants of the Composite Human Antibody™ anti-CLEVER-1 was selected and analyzed using iTope™ technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al., 2008) and using ICED™ (Cell epitope database). Petition 870250108080, dated 11 / 26 / 2025, page 48 / 116 43 / 50 T) of T cell epitopes related to known antibody sequences (Bryson et al., 2010). Sequence segments that were identified as significant non-human germline sequence ligands for human MHC class II or that had significant hits against TCED™ were discarded. This resulted in a reduced set of segments, and the combinations of these were re-analyzed, as above, to ensure that the junctions between the segments did not contain potential T cell epitopes. The selected segments were then combined to produce V-region heavy and light chain sequences for synthesis.For anti-CLEVER-1, four VH chains, VH1, VH2; VH3 and VH4 [SEQ ID NO: 13, 15, 17 and 19 (base sequence) and NO: 14, 16, 18 and 20 (amino acid sequence), respectively] and five Vk chains, VK1, VK2, VK3, VK4 and VK5 [SEQ ID NO: 21, 23, 25, 27 and 29 (base sequence) and NO: 22, 24, 26, 28 and 30 (amino acid sequence), respectively] were designed. The CDRs of the heavy chain VH1, VH2, VH3 and VH4 extend from base 91 to 111, 154 to 201 and 298 to 333; The light chain CDRs VK1, VK2, VK3, VK4, and VK5 extend from base 70 to 105, 151 to 171, and 268 to 294. The definitions of CDRs and protein sequence numbering are in accordance with Kabat. It is noteworthy that three of the VH chains have the N-linked glycosylation site potential removed (VH2, VH3, and VH4), and two of the VK chains have the unpaired cysteine ​​removed (VK4 and VK5). Example 8 Construction of Composite Human Antibody™ Variants

[00126] All variant genes of the VH and Vk regions of Composite Human Antibody™ for anti-Clever 1 were synthesized using a series of overlapping oligonucleotides that were paired, ligated, and amplified by PCR to provide the entire synthetic V regions. The assembled variants were then directly cloned into Petition 870250108080, dated 11 / 26 / 2025, page 49 / 116 44 / 50 Antitope pANT expression vector system for IgG4 VH and Vk chains (S241P) (Figure 7). The VH region was cloned using the Mlul and Hind III restriction sites, and the Vk region was cloned using the BssHII and BamHI restriction sites. All constructs were confirmed by sequencing.

[00127] Figure 7 shows the diagram of the pANT Antitope vector. Both Vh and VK vectors contain genomic DNA fragments incorporating introns and poly A sequences. The expression of both strands is controlled by a promoter and CMV selection (in the heavy chain vector) via a DHFR minigene. Example 9 Antibody Construction, Expression, and Purification

[00128] All combinations of VH and Vk chains of compound IgG4(S241P) (i.e., a total of 20 pairs) were stably transfected into NSO cells by electroporation. Stable transfections were selected using 200 nM methotrexate (MTX) (Sigma cat. no. M8407), methotrexate-resistant colonies for each construct were tested for IgG expression levels using an IgG4 ELISA, and the best expression lines were selected, expanded, and frozen in liquid nitrogen. Successful transfection and selection of stable clones were achieved for all variants except VH3A / Vk3 and VH4A / k3.

[00129] Composite anti-CLEVER-1 variants were purified from cell culture supernatants on a Protein A Sepharose column (GE Healthcare cat. no. 110034-93), buffer exchanged in PBS, and quantified by OD280nm using an extinction coefficient (Ec (0.1%) = 1.55) based on the predicted amino acid sequence. The main Composite Antibody™ variants were analyzed by reduction with SDS-PAGE. The corresponding bands Petition 870250108080, dated 11 / 26 / 2025, page 50 / 116 45 / 50 of the predicted sizes of the VH and Vk chains were observed without evidence of any contamination (Figure 8).

[00130] Figure 8 illustrates a Coomassie blue-stained SDS-PAGE gel of purified antibodies with selected protein A. 2 µg of each sample were loaded onto a NuPage 4-12% Bis-Tris gel (Invitrogen cat. no. NP0322BOX) and run at 200 V for 35 min. The size marker is a standard pre-labeled Fermentas PageRuler protein (cat. no. SM1811). Example 10 Linking Composite Human Antibodies™ to CLEVER-1

[00131] The binding of NSO-derived 3-372 composite antibodies to CLEVER-1 was evaluated by competitive ELISA. Dilution series of chimeric and composite 3-372 antibodies (5-0.078 µg / mL) were premixed with a constant concentration (0.6 µg / mL) of biotinylated mouse 3-372 antibody. These were incubated for 1 hour at room temperature in a 96-well Immulon Maxisorp plate (Fisher Cat. No. DIS-971-030J) pre-coated with 1 pg / mL of CLEVER-1. The binding of biotinylated mouse 3-372 to CLEVER-1 was detected using Streptavidin-HRP (Sigma Cat. No. S5512) and TMB single-solution substrate (Invitrogen Cat. No. 002023). The reaction was stopped with 3M HCl, the absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader, and the binding curves were plotted. The IC50 values ​​for each antibody were calculated and normalized to the IC50 of the chimera that was included in each respective ELISA plate.

[00132] The IC50s obtained show that several Composite Human Anti-CLEVER-1 Antibodies™ have better binding to CLEVER-1 than the chimeric 3-372. Competition data for the main variants are shown in Figure 9. Table 5 Characterization of Composite Bonding Human antiPetition 870250108080, dated 11 / 26 / 2025, page 51 / 116 46 / 50 CLEVER-1 Antibodies™ Region V IDs IC50 relative CH / CK 1.0 VH1 / VK1 0.84 VH1 / VK2 1.37 VH1 / VK3 1.63 VH1 / VK4 1.17 VH1 / VK5 1.13 VH2 / VK1 0.82 VH2 / VK2 0.95 VH2 / VK3 0.7 VH2 / VK4 0.79 VH2 / VK5 0.52 VH3 / VK1 0.76 VH3 / VK2 0.51 VH3 / VK3 - VH3 / VK4 0.47 VH3 / VK5 0.42 VH4 / VK1 1.86 VH4 / VK2 0.9 VH4 / VK3 - VH4 / VK4 1.2 VH4 / VK5 0.46

[00133] The relative IC50 was calculated by dividing the value of the test antibody by that of the chimera analyzed on the same plate. Example 11: Antibody binding in vitro

[00134] Human peripheral blood monocytes from healthy donors were collected and enriched from approximately 9 mL of peripheral blood by Ficoll gradient centrifugation. In Petition 870250108080, dated 11 / 26 / 2025, p. 52 / 116 47 / 50 cells are then plated in 96-well low-adhesion plates at a density of 1.2 x 10⁶ cells / well in IMDM medium supplemented with 1% human AB serum. The cells were treated with 1 pg / mL or 10 pg / mL of anti-CLEVER-1 3-372 antibody (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 21, 2001) or VH3 / VK5 (a humanized anti-CLEVER-1 antibody according to the present invention, recognizing said specific CLEVER-1 epitope) for 48 hours. HLA-DR expression was determined from CD14-positive cells after 48 hours using Fortessa LSR flow cytometry. Dead cells were excluded from the analysis based on a positive signal for the cell viability dye 7-AAD.

[00135] Human IgGs were used as a reference.

[00136] Figure 10A shows results of the determination of HLA-DR expression from CD14-positive cells. HLA-DR expression in CD14-positive cells increased with treatment with humanized anti-CLEVER-1 VH3 / VK5 antibody compared to the human IgG reference.

[00137] No difference in cell viability between treatments was observed. Therefore, it can be concluded that antibodies directed against CLEVER-1 do not affect monocyte survival. Example 12: TNF-α Measurement

[00138] Human peripheral blood monocytes from healthy donors were collected and enriched as described in Example 11. Monocytes from 3 mL of blood treated with red blood cell lysis buffer were allowed to adhere overnight in 6-well plates, washed once with PBS, and cultured for 3 days with 10 pg / mL of anti-CLEVER-1 3-372 or AK-1 antibody.

[00139] Soluble TNF-alpha was measured from the culture medium. Petition 870250108080, dated 11 / 26 / 2025, page 53 / 116 48 / 50 using a commercial ELISA kit for TNF-alpha (Invitrogen). The measurement results are shown in Figure 10B. Increased TNF-alpha secretion was observed in samples treated with anti-CLEVER-1 antibody compared to untreated samples or control samples treated with AK-1. Example 13: Syngeneic cancer models in mice

[00140] Established mouse mammary carcinomas E0771 were treated with 5 mg / kg of anti-CLEVER-1 (mStabl) or control isotype every 3 to 4 days until tumors reached a size of 1 mm3. The effect of anti-CLEVER-1 treatment on the recruitment and phenotype of TAMs, different monocyte subsets, and tumor-infiltrating leukocytes were evaluated using flow cytometry.

[00141] Figure 11A shows TAM repolarization in syngeneic E0771 breast carcinomas after administration of an antibody that binds to CLEVER-1. TAM repolarization is measured by the increase in macrophage populations expressing MHCII (in human HLA-DR) by flow cytometry. Each point represents the percentage of MHCIIhighCD11b+F4 / 80+ TAMs in a mouse. Tumors treated with anti-CLEVER-1 showed a similar level of TAMs (CD11b+F4 / 80+) compared to control-treated tumors. However, the TAM population in anti-CLEVER-1 tumors consisted of more pro-inflammatory macrophages (Ly6CloMHCIIhi) with lower expression of the type II marker, CD206.

[00142] Anti-CLEVER-1 treated TAMs secreted significantly more TNF-alpha compared to IgG-treated TAMs, as shown in Figure 11B. Each dot represents TAMs isolated from one mouse. Consistent with this, a decrease in FoxP3+ tumor infiltrating leukocytes was also observed.

[00143] The results indicate that CLEVER-1 is a potential target. Petition 870250108080, dated 11 / 26 / 2025, page 54 / 116 49 / 50 for macrophage-targeted immunotherapy. Example 14

[00144] As denoted in Example 1, antibodies 9-11 and 3-372 bind to distinct epitopes on human CLEVER-1, and the effects of this difference on signaling in human peripheral blood monocytes have now been studied. Figure 12 illustrates that CLEVER-1 binding with antibodies 9-11 and 3-372 promotes opposite effects on mTOR (mechanistic target of rapamycin) and c-Jun signaling in human peripheral blood monocytes.

[00145] Figure 12A shows a flow cytometry analysis of 9-11 and 3-372 binding in human CD14-positive monocytes (n = 2 donors, D1 and D2).

[00146] Figure 12B shows the results when the Human Phosphokinase Assay (R&D) was used to measure the activation of phospho proteins in CD14-positive cells (enriched by negative selection) after a 10-minute treatment with 20 μg / mL of 9-11 and 3-372. Phospho signals were normalized to cells treated with relevant isotype controls, ratIgG2a for 9-11 and mouse IgG1 for 3-372. As shown in Figure 12B, the 9-11 and 3-372 antibodies promote opposing effects on mTOR and c-Jun signaling in human peripheral blood monocytes. It is known that the mTOR pathway regulates macrophage polarization and the phenotype of immunosuppressive macrophages depends on c-Jun phosphorylation, where the results indicate that antibody 3-372 activates macrophages to change their phenotype from M2 macrophages to M1 macrophages. Other preferred options

[00147] It will be recognized that the agent capable of binding to human CLEVER-1, such as an antibody, the single-chain Fv or Fab fragment(s), the peptide(s), the macromolecule(s) and single-chain Fv or Fab fragment(s) of the humanized antibody and the compositions Petition 870250108080, dated 11 / 26 / 2025, p. 55 / 116 50 / 50 pharmaceuticals of the present invention can be incorporated in a variety of embodiments, only some of which are disclosed in this application. It will be apparent to those skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the embodiments described are illustrative and should not be considered restrictive. Petition 870250108080, dated 11 / 26 / 2025, p. 56 / 116

Claims

1 / 2 CLAIMS 1. Humanized antibody or single-chain Fv or Fab fragment capable of binding to a human Common Lymphatic Endothelial and Vascular Endothelial Receptor-1 (CLEVER-1) epitope, characterized in that said humanized antibody or single-chain Fv or Fab fragment comprises a) constant regions of the heavy chain and kappa light chain of human IgG4, and b) the combination of variable regions of the heavy and light chain of human IgG selected from the group consisting of the following combinations: SEQ ID NO: 14 and SEQ ID NO: 22; SEQ ID NO: 16 and SEQ ID NO: 22; SEQ ID NO: 16 and SEQ ID NO: 24; SEQ ID NO: 16 and SEQ ID NO: 26; SEQ ID NO: 16 and SEQ ID NO: 28; SEQ ID NO: 16 and SEQ ID NO: 30; SEQ ID NO 18 and SEQ ID NO: 22; SEQ ID NO: 18 and SEQ ID NO: 24; SEQ ID NO: 18 and SEQ ID NO: 28; SEQ ID NO: 18 and SEQ ID NO: 30, SEQ ID NO: 20 and SEQ ID NO: 24; and SEQ ID NO: 20 and SEQ ID NO:

30.

2. Humanized antibody or single-chain Fv or Fab fragment, according to claim 1, characterized in that the sequence combination of the variable region of the heavy chain and the light chain of human IgG are selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 30, SEQ ID NO: 18 and SEQ ID NO: 30 and SEQ ID NO: 20 and SEQ ID NO:

30.

3. Humanized antibody or single-chain Fv or Fab fragment, according to claim 1 or 2, characterized in that the constant region of the human IgG4 heavy chain and kappa light chain comprises the L248E and / or S241P mutations.

4. Humanized antibody or single-chain Fv or Fab fragment, according to any of the preceding claims 1 to 3, characterized in that it is for use in the treatment of cancer or chronic infections.

5. Humanized antibody or single-chain Fv or Fab fragment, according to claim 4, characterized in that it is for use in the treatment of cancer by reducing malignant tumor growth in an individual; and / or inhibiting the transmigration of cancer cells and the formation of metastases.

6. Pharmaceutical composition, characterized in that it comprises a humanized antibody or single-chain Fv or Fab fragment as defined in any of claims 1 to 3 and an appropriate excipient. Petition 870250108080, dated 11 / 26 / 2025, p. 58 / 116

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